Spencer David Balay
Transcript of Spencer David Balay
Cryptochrome Expression in the Zebrafish Retina: Potential Implications for Magnetoreception
by
Spencer David Balay
A thesis submitted in partial fulfillment of the requirements for the degree of
Master of Science
in
Physiology, Cell, and Developmental Biology
Department of Biological Sciences University of Alberta
© Spencer David Balay, 2018
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ABSTRACT
A variety of organisms have been shown to use the Earth’s magnetic field to orient in
local-spaces and to navigate long-distances. Although behavioural evidence of
magnetoreception has been reported in a diverse range of taxa, the proximate mechanisms
of this phenomenon have yet to be revealed. Some animals such as birds, appear to use a
light-dependent radical-pair-based magnetic compass. Ancient, light-sensitive proteins
called Cryptochrome (Cry) are currently the only known molecule found in vertebrates to
create radical-pairs, and thus are putative receptors. Cry is associated with the visual system
where it is co-localized with both short- and long-wavelength retinal cone photoreceptors in
adult birds, and therefore well-suited for light-dependent magnetoreception. Unfortunately,
due to the molecular inaccessibility of the avian model, Cry-cone interactions have seldom
been manipulated, and the requirement of Cry for magnetoreception has yet to be tested in
vertebrates. Additionally, Cry’s location in photoreceptors of other animals that display
magnetic behaviors is largely unknown.
This thesis utilized zebrafish (Danio rerio) to test if cry was associated with cones in
developing and adult fish retina. Zebrafish have six paralogs of cry and while most participate
in the circadian clock, the function of cry2 and cry4 are unknown. Here, I show that cry4 is
expressed in larval and adult zebrafish short-wavelength-sensitive (Ultraviolet-sensitive
(UV)) cones. Using nitroreductase (NTR)-mediated cell ablation and reverse transcription
quantitative-PCR (RT-qPCR), I found that cry4 expression decreased when UV cones were
ablated but was unaffected when neighboring blue cones were ablated in larval and adult
retina. cry2 did not appear to be expressed in UV cones and was unchanged after UV or blue
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cone ablation in both developmental stages. Although zebrafish magnetic behavior has only
been reported in adults, this work suggests larval fish may also have the molecular
framework for magnetoreception. While zebrafish are non-migratory, they can be used to
model other fish that migrate long-distances. Salmonids regenerate UV cones as they prepare
to migrate back to their natal streams for spawning. Currently, the functional significance of
this process has yet to determined. These findings could provide one explanation for this as
UV cones may enable magnetoreception via cry. In summary, I describe the localization of
cry4 in the zebrafish retina towards understanding whether fish have the molecular
mechanisms for light-dependent magnetoreception.
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PREFACE
This thesis is an original work done by Spencer D. Balay.
Approval for this study was obtained from the Animal Care and Use Committee:
Biosciences, under protocol AUP00000077.
Chapter 2 is modified from a manuscript submission to Current Biology, S. D. Balay
and W. T. Allison, “Exploring potential for light-dependent magnetoreception in zebrafish:
Cryptochrome expression in retinal photoreceptors.” This thesis abstract is modified from
the manuscript abstract in S.D Balay and W.T. Allison, “Exploring potential for light-
dependent magnetoreception in zebrafish: Cryptochrome expression in retinal
photoreceptors.”
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Dedicated to the late Dr. Fred Oscar Schreiber.
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ACKNOWLEDGEMENTS
I would like to thank my supervisor Dr. Ted Allison for his guidance, mentorship and
friendship throughout the duration of my thesis and time at the University of Alberta. I am
also extremely grateful for my Supervisory committee members: Dr. Colleen Cassady St.
Clair, for always lending an ear and giving me my first start in research and Dr. Douglas
Wylie, for welcoming me into his lab family and furthering my passion for visual
neuroscience. Thank you to Dr. Kimberley Mathot and Dr. Heather Proctor for sitting on my
examination committee. I am also indebted to Dr. Phil Oel, Dr. Michele Duval, Nicole Noel
and Emily Dong for introducing me to the Allison lab and for initiating the start of my
molecular training. I thank the current and former members of the Allison lab for making it
a such a warm work environment.
I would also like to thank Troy Locke, Jeff Johnston and Dr. John Phillips for their
technical and personal support throughout the duration of this thesis.
Thanks to the Department of Biological Sciences and the Faculty of Graduate Studies
for the funding support I received during this thesis.
I would like to especially thank my family for always being supportive throughout my
academic career. To June Schreiber, Dr. Gaylene Schreiber, Reg Wilkes, Kim Balay, Dave
Balay, Kevin Balay and Terry Kosabeck- you all have helped me grow exponentially. A very
special thank you to Sonya Widen, my life-partner, for constantly inspiring me to do and be
my best. Your words of wisdom have helped me more than you will ever know. I couldn’t
have reached this mountain top without you.
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TABLE OF CONTENTS
ABSTRACT ................................................................................................................................................ ii
PREFACE .................................................................................................................................................. iv
ACKNOWLEDGEMENTS ....................................................................................................................... vi
TABLE OF CONTENTS ......................................................................................................................... vii
LIST OF TABLES ..................................................................................................................................... ix
LIST OF FIGURES ................................................................................................................................... ix
LIST OF ABBREVIATIONS ................................................................................................................... xi
1 INTRODUCTION .............................................................................................................................. 1
1.1 The Mystery of Magnetoreception ............................................................................................... 1
1.2 Magnetic Fields ....................................................................................................................................... 2
1.3 Mechanisms of Magnetoreception ............................................................................................... 3
1.3.1 Magnetite Based Magnetoreception .......................................................................................... 3
1.3.2 Light-dependent magnetoreception .......................................................................................... 5
1.3.3 Radical-pair generation .................................................................................................................. 8
1.4 Cryptochrome and Light-Dependent Magnetoreception .............................................. 11
1.5 Zebrafish as a model for light-dependent magnetoreception .................................... 16
1.5.1 Zebrafish Cryptochromes ............................................................................................................ 17
1.5.2 Magnetoreception behavioral evidence and assays ......................................................... 19
1.5.3 Nitroreductase-mediated targeted ablation ........................................................................ 20
1.6 Purpose of study/ objectives ....................................................................................................... 21
2 EXPLORING POTENTIAL FOR LIGHT-DEPENDENT MAGNETORECEPTION IN
ZEBRAFISH: CRYPTOCHROME 4 IN A SELECT SUBTYPE OF RETINAL
PHOTORECEPTORS ............................................................................................................................. 29
2.1 Introduction ............................................................................................................................................... 29
2.2 Methods ........................................................................................................................................................ 30
2.1 Animal Ethics ....................................................................................................................................... 30
2.2 Zebrafish Maintenance .................................................................................................................... 30
2.3 Nitroreductase-Mediated Ablation ............................................................................................. 31
2.4 Riboprobe production and Fluorescent In-Situ Hybridization ........................................ 31
2.5 RNA Isolation ....................................................................................................................................... 32
2.5.1 Whole Larvae ............................................................................................................................... 32
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2.5.2 Adult Eye ....................................................................................................................................... 33
2.6 RT-qPCR ................................................................................................................................................. 33
2.6.1 cDNA synthesis ........................................................................................................................... 33
2.6.2 RT-qPCR Parameters ................................................................................................................ 34
2.6.3 Endogenous Control Stability Assay ................................................................................... 34
2.6.4 Primer Validation ....................................................................................................................... 34
2.7 Microscopy and Imaging and Figure Assembly ...................................................................... 35
2.8 Statistics ................................................................................................................................................. 35
2.3 Results and Discussion ............................................................................................................... 36
2.3.1 Expression of cry4 in larval zebrafish retina ....................................................................... 36
2.3.2 cry4 is specifically expressed in the UV cone subtype of larval zebrafish ................ 37
2.3.2 cry4 is expressed in adult zebrafish UV cones .................................................................... 39
2.3.3 Blue cone ablation does not measurably disrupt cry4 abundance in adult retina 40
2.3.4 cry4 and cone photoreceptors as potential magnetoreceptors in fish ...................... 41
3 GENERAL DISCUSSION AND FUTURE DIRECTIONS .............................................................. 53
3.1 The role of UV cones and cone mosaics in magnetoreception......................................... 53
3.1.1 UV vision may be suited for visually perceiving magnetic fields ................................. 55
3.1.2 Fine tuning of visually mediated magnetoreception may be accomplished by long-
wavelength photoreceptors .................................................................................................................. 57
3.2 cry2 and cry4’s function in zebrafish retina: magnetoreception, circadian photoreception or neither .......................................................................................................................... 59
3.3 Future Experiments to test LDRPM in Zebrafish .................................................................... 62
3.3.1 Behavioral experiments to test for LDRPM .......................................................................... 62
3.3.2 Genome editing of cry in zebrafish .......................................................................................... 65
3.3.3 Imaging magnetically dependent neuronal activity via Ca2+ ......................................... 67
3.4 Final Conclusions ..................................................................................................................................... 69
Literature Cited .................................................................................................................................... 72
APPENDIX A ........................................................................................................................................... 91
A.1 Generation of slc45a2 mutant zebrafish ..................................................................................... 91
A.1.1 sgRNA creation ........................................................................................................................................ 91
A.1.2 Injection process ..................................................................................................................................... 91
A.1.3 Genotyping via sequencing and RFLP............................................................................................. 92
A.1.4 Generation of crystal and crystal-based transgenics ................................................................ 93
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LIST OF TABLES
Table 1. Transgenic zebrafish lines used in experiments. ................................................................. 43
Table 2. Primers used for Cryptochrome riboprobe synthesis. ....................................................... 43
Table 3. RT-qPCR primers used for Cryptochrome mRNA quantification................................... 44
Table A1. Oligonucleotides used for sgRNA synthesis. ....................................................................... 94
Table A2. Primers used for ua5015 and ua5020 genotyping. .......................................................... 94
LIST OF FIGURES
Figure 1. The geomagnetic field. .................................................................................................................. 24
Figure 2. Model of light-dependent magnetoreception. ..................................................................... 26
Figure 3. Cryptochrome can create radical-pairs between FAD and Trp. .................................... 27
Figure 4. How the magnetic field may alter animals’ visual perception. ...................................... 28
Figure 5. Larval zebrafish UV cones express cry4 during the day................................................... 45
Figure 6. Ablation of UV cones, but not blue cones, drastically decreases cry4 in larval
zebrafish. ............................................................................................................................................................... 47
Figure 7. UV cone ablation decreases cry4 expression is adult zebrafish retina. ...................... 48
Figure 8. Effective blue cone ablation does not disrupt cry4 expression in adult zebrafish
retina. ...................................................................................................................................................................... 49
Figure 9. cry2 expression is not changed significantly after both UV cone and blue cone
ablation in larval and adult zebrafish......................................................................................................... 50
Figure 10. No cry4 probe control for double fluorescent in situ hybridization on adult retina.
................................................................................................................................................................................... 51
Figure 11. β -actin is a suitable endogenous control for RT-qPCR experiments. ...................... 52
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Figure 12. Molar absorption spectra of FAD in different Cry redox states. ................................. 71
Figure A1. Larval nacre zebrafish injected with slc45a2 CRISPR mix show mosaic pigment
defects. ................................................................................................................................................................... 95
Figure A2. ua5015 (slc45a2-/-; mitfa-/-) have no pigment in the RPE and can be observed
through development. ...................................................................................................................................... 96
Figure A3. Pigmentless ua5020 mutants are generated from crossing ua5015 to casper. ... 98
Figure A4. Tgua5020(sws1:nfsb-mCherry; sws2:GFP) transgenics allow easy visualization of
fluorescent reporters in zebrafish retina. ................................................................................................ 99
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LIST OF ABBREVIATIONS
µl microlitres
µM micromolar
µs microseconds
Ca2+ calcium
CaMPARI calcium modulated photoactivatable ratiometric integrators
Cas CRISPR associated protein
CRISPR clustered regularly interspaced short palindromic repeat
cry or Cry cryptochrome
DMSO dimethyl sulfoxide
dpf days post fertilization
FAD flavin adenine dinucleotide
FADH− anionic fully reduced state of FAD
FADH• neutral semiquinone radical of FAD
GCL ganglion cell layer
GECIs genetically encoded calcium integrators
HC horizontal cells
hpf hours post fertilization
INL inner nuclear layer
LDRPM light-dependent radical-pair magnetoreception
lws1 long-wavelength sensitive 1 (opsin)
lws2 long-wavelength sensitive 2 (opsin)
MBM magnetite-based magnetoreception
MHz megahertz
ms millisecond
MS-222 tricaine methanesulfonate
MTZ metronidazole
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nl nanolitre
nm nanometers
nT nanotesla
NTR nitroreductase
ONL outer nuclear layer
PCR polymerase chain reaction
PTU 1-phenol-2-thiourea
RGC retinal ganglion cell
rh1 rhodopsin
rh2-1 medium-wavelength sensitive 1 (opsin)
rh2-2 medium-wavelength sensitive 2 (opsin)
rh2-3 medium-wavelength sensitive 3 (opsin)
rh2-4 medium-wavelength sensitive 4 (opsin)
RP radical-pair
RPE retinal pigmented epithelium
RT-qPCR reverse transcription quantitative polymerase chain reaction
SFAD*/FADox neutral fully oxidized state of FAD
SFAD•− singlet anionic semiquinone radical of FAD
sws1 short-wavelength-sensitive 1 (opsin)
sws2 short-wavelength-sensitive 2 (opsin)
TFAD•− singlet anionic semiquinone radical of FAD
Tg transgenic
Trp tryptophan
UTR untranslated region
UV ultraviolet
ZT zeitgeber
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1 INTRODUCTION
1.1 The Mystery of Magnetoreception
Magnetoreception is argued to be one of the most complex and intriguing sensory
pathways yet to be fully described. Consider it in relation to the understanding of much more
palpable senses such as the visual perception of light, which is something humans experience
everyday. We have a relatively firm grasp on the development and evolution of
photosensitive organs, have identified countless receptors with specific functions, know
detailed neural pathways in many animals, and can predict and treat related diseases. The
stimuli (visible light) has certain properties we have long been aware of: it cannot pass
through certain solid objects (like your arm), it acts like a particle and a wave and its tangible
presence can be experienced with the flick of a switch or the strike of a match in a darkened
room. Despite the centuries of progress made to understand vision, many things about this
“well-described” sense remain unknown.
Now consider the perception of magnetic fields: a completely intangible experience to
the conscious human sensory system. Nevertheless, this sense has been shown to exist in
plants, many invertebrates and every major class of vertebrate. In this case, even the mere
identification of magnetoreceptive organs cannot be made with certainty. Why is so little
known about this apparently common occurrence? The stimuli here, can pass through any
biological tissue, is present in almost every situation of our lives, and has been around for
more than 4 billion years [1]. How animals detect this omnipresent entity has intrigued
scientists for decades. Regardless, much progress has been made towards understanding the
mysteries of magnetoreception and is briefly reviewed below.
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1.2 Magnetic Fields
The magnetic field of the Earth can be most simply conceptualized akin to a bar magnet
(Figure 1). It is dipole in nature such that it produces two magnetic poles closely positioned
to the geographical poles of the Earth. This configuration permits several unique magnetic
characteristics to exist, commonly described as declination, inclination and intensity.
Declination is the angle that varies between geographic and magnetic poles, while inclination
is the angle that the magnetic field makes with the Earth’s horizontal; at the magnetic North
pole, the magnetic field points directly down, (inclination=+90⁰). This angle decreases as it
approaches the Equator (inclination=0⁰) and becomes negative (points up) as it reaches the
South pole (inclination=-90⁰). Similarly, intensity of the geomagnetic field exists in a
gradient, where it is strongest at the poles (~60,000 nT) and weakest at the Equator
(~30,000 nT) [2]. Despite some variation due to secular drift or anomalies created by
magnetic storms and enriched deposits of iron in the crust of the Earth, the geomagnetic field
acts as a reliable, constant spatial grid. This entity exists in a stable state regardless of
weather, temperature or time of day, and has been shown to aid a variety of animals in
orientation and navigation behaviors [3].
All major classes of vertebrates (fish, amphibians, reptiles, birds and mammals; [3-9])
and some invertebrate classes (molluscs, insects and crustaceans; [10-14]) have been shown
to use magnetoreception for a suite of spatial behaviors. Magnetically derived compass (used
to determine direction) and map (used to determine geographical position) information aids
in animal homing, migration, predation and exploration of local environment [4,8,9,15-26].
Although a wealth of behavioral evidence for magnetoreception exists across a diverse range
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of taxa, the underlying molecular, neural and physiological mechanisms of this phenomenon
remain unknown. So far, two main magnetoreception hypotheses have predominately been
suggested: magnetite-based magnetoreception (MBM; [27-29]) and light-dependent radical-
pair magnetoreception (LDRPM; [30-36]). These two mechanisms appear to fulfill different
roles in different animals and are not necessarily mutually exclusive; indeed, some animals
such as birds, rodents, salamanders and fish display evidence of multiple modes of
magnetoreception [37-40]. Generally, MBM is thought to mediate map sense, while LDRPM
has been suggested to mediate compass sense. As such, the molecular underpinnings, neural
pathways and behavioral outputs appear to be separate between MBM and LDRPM and are
outlined in the following section.
1.3 Mechanisms of Magnetoreception
1.3.1 Magnetite Based Magnetoreception
MBM involves inherently magnetic particles that are integrated into a sensory
pathway; that is, they are physically coupled to ion channels in a sensory cell (for review see
[27,29,41]). Mechanical force exerted on the particles by changing magnetic intensity or
direction could be used to transduce a biologically relevant signal by opening or closing ion
channels [42,43]. This type of magnetoreception would theoretically allow magnetic North
to be distinguished from magnetic South relative to the position of the navigator (i.e.
provides a map sense). This system exploits small intensity and potentially inclination
changes in the magnetic field and does not require light to function [44]. This mechanism has
been supported by the discovery of magnetite, an iron oxide derivative, in magnetotactic
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bacteria [45]. Since its initial discovery, magnetite has been proposed to be found in a variety
of animals such as bees, birds and fish [46-50]. Although a number of studies claim to have
found magnetic material in magnetoreceptive animals, its presence in biological tissue
remains controversial [51,52].
Notably, MBM is the most popular hypothesis for fish magnetoreception [41,49,53].
The olfactory system appears to be involved with MBM, as electrophysiological recordings
from the trigeminal nerve of rainbow trout (Oncorhynchus mykiss) [49] and birds [54] show
altered electrical output when the intensity of the magnetic field is changed. Magnetite has
also been proposed to be found in trout olfactory lamellae and throughout the adult zebrafish
[47,48,50]. The inactivation of trout’s ophthalmic branch of the trigeminal nerve eliminates
a physiological response to magnetic fields [55], supporting its role in magnetoreception.
Some fish behavior also supports this mechanism: migratory Pacific salmon appear to have
a map sense, where they swim along magnetic intensity and inclination field lines similar to
their natal streams [21,23]. Additionally, zebrafish, medaka (Oryzias latipes), Chinook
salmon (Oncorhynchus tshawytscha) and rainbow trout have been shown to be responsive to
magnetic fields in the dark, which supports a light-independent based magnetoreceptor
[40,55-60], similar to what is found in subterranean mole rats (Spalax ehrenbergi) and bats
[6,61,62]. Other marine migrants, such as turtles, appear to have a magnetic map that is
sensitive to the intensity and inclination of magnetic fields [63-65]. Interestingly, although
magnetite is thought to mediate this behavior [63,66], application of radio-frequency fields
(a diagnostic tool for LDRPM) has been shown to disrupt juvenile snapping turtle (Chelydra
serpentina) alignment [67], which supports a light dependent radical-pair magnetoreception
pathway (described in section 1.3.2). Additionally, salmon, zebrafish and medaka show
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different behavioral responses to magnetic fields in conditions with light and without light
[5,40,56-59]. These findings, among others, suggest that fish and other animals likely contain
another mechanism of magnetoreception that is light-dependent.
1.3.2 Light-dependent magnetoreception
Light-dependent magnetoreception has been characterized in a variety of animals
including birds, amphibians, rodents and insects [4,12,14,16,19,37,38,68-71]. The first step
in this mechanism (described in section 1.3.3) involves the detection of light, rather than
processing of magnetic information. In agreement with this, orientation ability of animals
that exhibit light-dependent magnetic behaviors appear to be affected by intensity,
wavelength and polarization of light. The most compelling evidence comes from the affect of
wavelength on orientation ability seen in migratory birds and salamanders. Eastern red
spotted newts (Notophthalmus viridescens) trained to orient under full spectrum light (≥450
nm) show a 90⁰ rotated orientation when tested under monochromatic light with
wavelengths above 500 nm [4,16,37]. Many birds such as : European robins (Erithacus
rubecula) garden warblers (Sylvia borin), chicken (Gallus gallus), Australian silvereyes
(Zosterops lateralis) and carrier pigeons (Columba livia domestica) exhibit magnetoreceptive
behaviors and appear to have a magnetic compass that operates best in short-wavelength
light (370 nm to 570 nm; for review see[69]). When tested under wavelengths of 590 nm
and above, compass orientation is abolished, and normally preferred headings are
randomized. Even animals extremely divergent to birds, such as fruit flies (Drosophila
melanogaster) [11] and the American cockroach (Periplaneta americana) [14], show a
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magnetic behavior that is present under short-wavelength light but is disrupted under long-
wavelength light.
In cockroaches, increasing the intensity of light permits this behavior to manifest in
longer-wavelengths that normally do not allow the orientation behavior. This “behavioral
rescue” is only observed up to a certain wavelength, where even extremely high intensities
of light cannot force the behavior above 505 nm [14]. Additionally, intensity of light appears
to alter orientation abilities in birds such as European robins [68,72-74]. The response to
increased intensities of different wavelengths of monochromatic light causes a suite of
behaviors including disorientation, polar orientation to magnetic North and rotated
orientation in the East-West axis. Why these different responses were exhibited is currently
unknown, but these findings suggest that photoreceptors, specifically those tuned to precise
wavelengths, are likely important for magnetoreception. It is also proposed that polarized
light may interact with the magnetic compass in zebra finches (Taeniopygia guttata). When
finches were trained to polarized light cues that are parallel to magnetic field direction (or
vice versa) magnetic orientation abilities were intact. When either the magnetic field or
direction of polarized light are changed to be perpendicular in respect to each other,
orientation abilities were randomized [75]. Although how (and if) polarized light and
magnetic fields interact is currently under debate [76], light-dependent magnetoreception
appears to be well-established in a variety of organisms.
Due to these findings and others, it is thought that light-dependent magnetoreception
would most favourably be mediated in light-sensing organs such as the pineal (as seen in
some vertebrates) or the retina. The pineal appears to play a role in salamander orientation
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[77,78] and has been implicated as magnetosensitive in birds [79], but its magnetoreceptive
role in other vertebrates who display this structure (fish and reptiles) is unknown. In birds,
although the pineal can respond to magnetic fields, it is not necessary for magnetoreception
[80,81], pointing towards the involvement of the retina. Indeed, neuronal areas such as the
nucleus of the basal optic root (nBor) of pigeon accessory optic system show
electrophysiological changes to magnetic fields, suggesting retinal input aids in
magnetoreception [82]. Also, the optic tectum has been described to show
electrophysiological responses to magnetic fields [79], but these results remain
controversial [83]. From a neurological perspective, the most compelling evidence of visually
mediated magnetoreception involves experiments manipulating Cluster N in the Visual
Wulst of birds [84]. Cluster N receives projections from retinal ganglion cells (RGCs) via the
thalamofugal pathway and is highly active during magnetic compass orientation [85-87].
Lesion of Cluster N disrupts magnetic orientation abilities, but other navigational means (of
sun and star compasses) remain fully functional, suggesting magnetic information is
specifically processed in Cluster N and transduced via pathways that originate in the retina
[84]. The hemispherical shape of the eye also may serve an important magnetoreceptive
purpose, specifically the ordered array of cone/rod photoreceptors along the back of the
retina [36,88-90]. This layout allows potential magnetoreceptors to receive magnetic
information across various angles and orientations in respect to the animal. Additionally, the
semi-fixed position of photoreceptors has historically been thought to be required for
radical-pairs to function as directional magnetoreceptors [33,36,88-91]. The details of how
these photoreceptors may be sensitive to magnetic fields, is outlined below.
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1.3.3 Radical-pair generation
The central theory behind LDRPM involves the creation of magnetically sensitive
radical-pairs1 after a photochemical reaction. This biophysical model was first proposed by
Schulten in 1978 [30,35], and since then has become one of the leading hypotheses of
magnetoreception (for review see [33]). In LDRPM, light-induced electron transfer is
responsible for creating radical-pairs. These radicals are magnetic in nature, due to a
magnetic moment created by the quantum spin state of each unpaired electron. Spin state is
dependent on the effect of the internal magnetic field, caused by the rotation of nearby nuclei
(hyperfine interactions). After light absorption causes an electron to transfer from a donor
to an acceptor, the spin of the radical-pair begins in a singlet-state, with each unpaired
electron spinning in opposite orientations (or antiparallel; Figure 2C). Over time, hyperfine
interactions cause the spins of the unpaired electrons to oscillate such that their orientations
change with respect to each other. Eventually, the spin of the two unpaired electrons in each
radical end up parallel (triplet-state) to each other [36].
After initial radical-pair formation (singlet-state), electrons usually back transfer to
the more favourable ground-state and abolish the radical-pair (i.e. forbid the formation of
the triplet-state). The radical-pairs that do proceed to the triplet-state, forbid the electron
back transfer, and increase the life time of the active radical-pair. An external magnetic field
(such as the geomagnetic field) can alter the interconversion rate of single-to-triplet radicals
1 A radical is a molecule with an odd number of electrons. Radical-pairs are two radicals that are created simultaneously after some energy-level splitting stimulus.
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by influencing how the electrons oscillate in relation to each other (Zeeman interactions)2.
The ratio of the products created from each radical-pair reaction (i.e. chemical signals
produced from either singlet or triplet-state) could be compared and cause the molecule to
act differently depending on which state predominated in a certain scenario (Figure 2). If the
triplet-state is favoured, where the ground-state promoting electron back transfer is
prohibited, the molecule could theoretically spend more time in a signalling form. In LDRPM,
the bias towards triplet-state is ultimately influenced by the presence of the geomagnetic
field thus creating a biological magnetic sensor [32,33,36].
The direction of the geomagnetic field can also influence this process: the external
magnetic field differentially affects interconversion rates based on its direction with respect
to the radicals (i.e. not every radical will be influenced the same way). This anisotropy is
what allows the magnetic field/ radical-pair interaction to actually provide directional
information from magnetic fields (i.e. provide a compass sense), rather than just simply
detect the presence or absence of magnetic fields, which would occur if all radicals reacted
in the same fashion to external magnetic fields [33,36,88,89,92]. Indeed, a variety of studies
have shown that geomagnetically relevant magnetic fields alter singlet-triplet
interconversion rates of radical-pairs in vitro [93-96] and specifically the direction of the
magnetic field can change the yield of radical-pair products, providing evidence that a
chemical compass is biologically feasible [97].
Besides theoretical and recent experimental data, other diagnostic experiments have
been developed to test if radical-pairs are involved in magnetoreception. Potentially the
2 So long as the radical-pair intermediates persist long enough for the Earth’s magnetic field to interact with them (longer than 1µs; [33,92]).
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most compelling experiment is done by exposing orienting animals to low level radio
frequency fields [36,98,99]. It is known that an isolated electron in a radical-pair (void of
hyperfine interactions) spins at a well-characterized frequency (the Lamar frequency,
around 1.4 MHz depending on location) in an ambient magnetic field. Application of
frequencies that encompass the energy-level splitting required to switch radical-pairs from
singlet to triplet states (1.4 MHz) can alter the interconversion process, and ultimately
disrupt the effect of the external magnetic field (for review see [33]). In vitro, it has been
shown that 1-100MHz frequencies disrupt radical-pair interconversion [100]. More
convincingly, European robins that were exposed to broadband (0.1-10MHz) or discrete
(1.3MHz) frequencies were disoriented when being tested for magnetic compass [98,101].
Additionally, low levels of anthropogenic electromagnetic noise (non-uniform radio
frequencies), and weak broadband frequencies have been shown to disrupt orientation in
birds, illustrating the range of this sensitivity [102,103] . Orientation of other animals such
as garden warblers, zebra finches and cockroaches [104-106] also all appear to be disrupted
by low-level radio frequencies, supporting a LDRPM mechanism. Interestingly, it has been
shown that if juvenile turtles are acclimated in radio frequencies parallel to the magnetic
field, they are able to exhibit a spontaneous magnetic alignment that is abolished when the
radio frequency field is turned off [67]. This suggests some plasticity in the way radio
frequency fields can influence magnetoreception. There is much to learn about how radio
frequencies interact with radical-pairs (for review see [107]), but regardless, the idea that
radical-pairs are likely to function as a chemical compass has gained considerable ground in
the last two decades.
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For almost 20 years after the initial conception of LDRPM, the theoretical molecule
capable of radical-pair formation remained unidentified in animals that displayed light-
dependent magnetic behaviors. In 2000, Ritz et al., [36] suggested that Cryptochrome (Cry),
a recently characterized protein at the time [108], may fulfill the requirements as a light-
dependent magnetoreceptor, as it can form light-induced radical pairs, and is found in a
variety of organisms including vertebrates.
1.4 Cryptochrome and Light-Dependent Magnetoreception
Cry was first identified in plants as a “cryptic” blue light/UV-absorbing photoreceptor
that controls development and growth [108]. Evolutionarily, Crys are homologous to
photolyase, an ancient enzyme that catalyzes repair of UV-induced DNA damage in
prokaryotes and eukaryotes [109]. Most Crys have lost DNA repair activity but all have
retained two N-terminal domains from their photolyase ancestors; a photolyase domain and
a flavin adenine dinucleotide (FAD) binding domain which both bind chromophore cofactors
that are responsible for short-wavelength light absorption [110-112]. In photolyases and
Arabidopsis thaliana Cry, phototransduction is mediated by electron transfer between a triad
of Tryptophan (Trp) residues and the FAD cofactor [113,114]. In various animal Crys, the
position of these tryptophan’s is highly conserved[112] and therefore, the magnetically
sensitive radical pairs are proposed to be created between them and FAD (Figure 3). Recent
evidence suggests the favoured electron transfer pathway may not be required for
magnetically dependent Cry biochemical changes or magnetoreceptive behavior, but this
notion requires further investigation [13,115].
12
Crys also have a well-characterized role in tuning biological rhythms as part of the
circadian clock through both photic and aphotic mechanisms [110,116]. Crys photoreceptor
function depends on which functional group they belong to [112,117-119]. The functional
groups of Cry are commonly described as either: Type I Crys, which are directly light-
sensitive and seem to act as circadian photoreceptors in invertebrates, Type II Crys which
have no described light-dependent function and regulate transcription of clock genes in the
vertebrate circadian clock, and Cry-DASH which appears to have retained the ability of photo
repair but are mostly considered as intermediates between Photolyases and Cry. CryDASH
is found in a unique subset of organisms such as plants, cyanobacteria, insects and zebrafish,
and little is known about their potential role in magnetoreception (for review see [110]).
Crys’ magnetoreceptive function is largely built off biochemical models describing the
creation of magnetically sensitive radical pairs via redox reactions from Arabidopsis and
Escherichia coli [95,120]. Stereotypically, after light is absorbed by the FAD co-factor,
electron transfer reduces fully oxidized FAD to form the radical semiquinone form (SFAD* →
SFAD•−) and oxidizes the first surface exposed TrpH, to form the radical-pair S[FAD•− TrpH•+],
which is conserved in singlet form. From the singlet-state, several things may occur: 1)
singlet-triplet interconversion via hyperfine and Zeeman interactions with the internal and
external magnetic field S[FAD•− TrpH•+] ↔ T[FAD•− TrpH•+ ], 2) the radical-pair can reverse
transfer it’s electron, regenerating the ground-state of the protein S[FAD•− TrpH•+] → FAD +
TrpH, or 3) the deprotonation of the radicals can give rise to a second radical-pair, whose
spin is proposed to not be amendable by magnetic fields S[FAD•− TrpH•+] or T[FAD•− TrpH•+ ]
→ [FADH• Trp• ] which then is slowly (~10 ms) brought back to the ground-state [33,95]. It
has been hypothesized that short-wavelength light permits the formation of the initial,
13
magnetically sensitive radical-pair to form (RP1 in Figure 3), while long-wavelength light
may influence the formation of the second radical pair (RP2, Figure 3). This wavelength-
dependent process could provide one reason why short and long-wavelength light appear to
affect animals’ magnetic compass antagonistically [121,122]. The different spectral
properties of FAD intermediates and how they may relate to magnetoreception are discussed
in Chapter 3 (Figure 12).
Aside from biophysical models and calculations, it has been recently shown that Cry4
has a high affinity for FAD and is thus suitable for light-dependent magnetoreception [123].
Most vertebrate Type II Crys (such as Cry1 and Cry2) have low binding affinity for FAD and
are probably not functional for radical-pair generation [123]. Cry4 is also only found in
animals that exhibit magnetoreceptive behaviors such as birds, amphibians, reptiles and fish
[112,117,119]. Cry4 from zebrafish and chicken have been shown to undergo blue light
dependent conformational changes and has been isolated with large amounts of FAD, making
it biologically suited for magnetoreception and light-dependent radical-pair generation
[123-126]. Also, Cry4 appears to be more closely related to photosensitive Type I Crys of
invertebrates [112,119], and from phylogenetic studies in fish, is predicted to have evolved
earlier than vertebrate and invertebrate Type 1/2 Crys, suggesting its form is the ancestral
state [117]. For these reasons, Cry4 appears to be best biochemically suited to be a candidate
molecular magnetoreceptor.
For functional support of Cry-based magnetoreception, when Type I Cry is expressed
ectopically in larval fruit fly motoneurons, application of a magnetic field increases action
potential firing, showing that Cry can influence neural activity [115]. In addition to
14
theoretical and electrophysiological data, behavioral experiments in both Drosophila and
cockroaches have shown that Cry is necessary for magnetoreceptive behaviors: flies without
Cry were unable to respond to a trained magnetic stimulus that wild-type flies could detect
[12]. Insertion of Type II Crys (such as the human ortholog Cry2), and butterfly Cry2 in Cry-
deficient flies was able to rescue light-dependent magnetoreception, showing that various
Cry types are sufficient for magnetoreception in the right biological system [13,71]. In
Drosophila, other behaviors such as geotaxis, seizure susceptibility and circadian clock
entrainment appear to also be modulated by Cry and magnetic fields [127-131].
Over the last two decades a wealth of theoretical, physiological, behavioral, and
molecular data has suggested that Cry participates in LDRPM of various animals. Despite this,
how magnetic signals are processed and interpreted remain unknown. LDRPM’s
requirement of light, and the magneto-relevant architecture of the eye make the visual
system likely involved. In support of this, Crys in every functional group have been shown
to be associated with the visual system of many different taxa. In sponges who lack opsins,
Cry is found in the pigmented eye ring and is thought to mediate phototaxis in free swimming
larval stages [132,133]. Cry is also found in the compound eyes of invertebrates such as fruit
flies and cockroaches, and is suggested to have roles in circadian regulation, sun-compass
tuning, and magnetoreception [12,14,134,135]. In zebrafish, multiple Crys are found in all
layers of the larval and adult retina, but their function and cellular location remain unknown,
and understudied [118,119,125,126]. In birds such as the garden warbler, Cry1b is in the
RGC’s, and inner segments of photoreceptors and is thought to play a circadian or
magnetoreceptive role [85,136,137]. Cry1a on the other hand, is localized to the outer
segments of European Robin and chicken UV cones and appears to be well-suited for light-
15
dependent magnetoreception [138,139] but see [123]. Recently, Cry4 has been described to
localize with single long-wavelength and double cones of the night-migratory European
robin and the non-migrating chicken, but currently its role in magnetoreception remains
unknown [124]. Even in some mammalian species, Cry is associated with the visual system:
Cry1 localizes to the short-wavelength photoreceptors, which are homologous to bird and
fish UV cones, of fox, bear, dog and orangutan [140]. In humans, Cry2 is localized in the RGCs
and appears to participate in light-independent circadian regulation [141].
Crys’ localization to cone photoreceptors is particularly interesting for
magnetoreception because it fulfills several requirements of LDRPM [36,69,89]. Anchoring
to the outer segments of UV or double cones allows some degree of fixation of one Cry protein
to relation to another, allowing the radical-pair outputs to be compared across the retina.
Also, if Cry is paired to a visually transducing receptor (opsin), it might exploit the
electrochemical pathway cones use to send signals to neuronal areas that process visual
information, allowing the magnetic field to alter the visual perception of an animal
[36,90,142]. Indeed, one of the leading ideas of the how magnetic fields may be perceived is
best described as a lined-spatial pattern which overlays an animals’ visual field (Figure 4).
The light and dark lines result from individual photoreceptor-Cry cells being activated or
inactivated, due to orientation with respect to the magnetic field (and ultimately the ratio of
the products generated by singlet or triplet radical-pairs). In birds, it is specifically thought
that the changing inclination is what alters their visual perception. If inclination is reversed
180⁰ (90⁰ to -90⁰), while the horizontal magnetic field is unchanged (mN=mN) migratory
birds reversed their orientation ~180⁰, illustrating that they are sensing the inclination
(poleward vs equatorward) rather than the polarity (North pole vs South pole) [143].
16
Inclination compasses have been suggested for other long-distance migrants such as turtles,
and short distance migrants such as newts, suggesting even small inclination changes could
alter magnetic field detection [37,144]. Regardless, magnetic fields could be manifested in
many undescribed ways. Currently, this is the leading model for how light-dependent
magnetic information is processed.
Although studied extensively, the molecular mechanisms of magnetoreception
continue to remain a mystery. The vertebrate light-dependent magnetoreception field has
historically lacked mechanistic experiments, where the necessity and sufficiency of
proximate mechanisms, such as receptors and neuronal areas are manipulated in a reliable
and repeatable way, as seen in the invertebrate literature. This calls for the use of a
vertebrate model that permits precise testing of molecular machinery but still possesses
magnetically-dependent behavioral outputs.
1.5 Zebrafish as a model for light-dependent magnetoreception
Zebrafish are an ideal model for studying molecular mechanisms of visually-mediated
behaviors: these teleost fish undergo external fertilization and are transparent at early
stages, which allows for easy monitoring of the developing visual system. By 51 hours post
fertilization (hpf) opsin expression is detectable, and by 5 days post fertilization (dpf)
zebrafish exhibit behavioral responses to light [145,146]. Additionally, zebrafish retina
continues to grow throughout the life of the fish and can robustly regenerate retinal neurons
after chemical, physical and light-induced damage [147-150], a characteristic that is found
in most teleosts [151,152] but is limited in other models such as mouse and birds [153].
17
The zebrafish eye is extremely anatomically, genetically and physiologically conserved
with other vertebrates. Zebrafish are tetrachromats that have four cone subtypes: UV (sws1),
blue (sws2), green (rh2-1, rh2-2, rh2-3, rh2-4) and red (lws1, lws2) cones that are defined by
their wavelength sensitive opsin. Additionally, rods (rh1) are low-light photoreceptors that
mediate dim and night vision, that are present in the zebrafish retina [154-156]. In
adulthood, cone photoreceptors are laid out in the retina as a precise mosaic with alternating
rows of UV and blue cones, parallel to rows of red and green double cones with rods packed
in between the cones. The mosaic is arranged so that the red cones flank the blue cones, and
the green cones flank the UV cones [157-161]. In the larval zebrafish, the row photoreceptor
mosaic is not as apparent, although they do contain stereotypical amounts of each
photoreceptor type relative to each other. As the fish grows, more photoreceptors are added
at the periphery of the retina and organized into the mosaic [161]. Although many animals
contain mosaics, their function is largely unknown. A potential role in magnetoreception is
discussed in Chapter 3.
Within the well-characterized visual system of zebrafish, many Cryptochromes have
been recently described [118,119,125]. Despite their presence, the specific functions and
cellular locations of Cry are not fully understood.
1.5.1 Zebrafish Cryptochromes
Members of the teleost lineage have the largest number of Crys present in modern
day genomes [112,117,119]. Zebrafish Cry has undergone three whole genome duplications
including the teleost specific genome duplication about 350MYA (for review see [162]). As
such, zebrafish possess seven distinct Crys named: cry1aa, cry1ab, cry1ba, cry1bb, cry2, cry4
18
and cry-DASH. The cry1-paralogs have been exclusively shown to participate in core
circadian regulation, which requires a nuclear localization sequence (NLS) and the ability to
bind other core circadian proteins such as BMAL1 [118,119,125]. Cry1aa appears to remain
light-sensitive but acts extremely similar to non-light sensitive Cry1/2 involved in
mammalian circadian regulation [118,125]. The function of cry2 and cry4 remain yet to be
identified. cry2 has evolved an NLS and is found in the nucleus and cytoplasm but cannot
bind BMAL1 and inhibit transcription related with circadian regulation. Additionally, cry4
does not have an NLS and even when forced into the nucleus is unable to bind to BMAL1,
suggesting it does not participate in core circadian regulation [118,125]. Zebrafish Cry4 has
also been isolated with relevant amounts of FAD and has been shown to undergo light-
dependent conformational changes, suggesting it could participate in light-dependent
magnetoreception [126].
The various crys show different expression patterns in zebrafish; in larval fish, all crys
are found broadly in the retina and the brain [118,119]. cry4 is specifically expressed in the
optic tectum and the intestine. In adult retina, cry expression seems to oscillate, in various
cell types: cry1 paralogs are highly expressed in most retinal layers during light time points,
and lowly expressed at night. Notably, cry2 has been suggested to be found in the outer
nuclear layer (ONL), specifically in the short-wavelength cones right before light is turned
on (Zeitgeber Time3, ZT=23). cry4’s expression seems to be limited to cone photoreceptors
in the ONL at around noon (ZT=4) and is broad in the ONL throughout the day, with
3 A Zeitgeber is an environmental cue used in the synchronization of animal’s clocks. In this case, light is the Zeitgeber. ZT refers to the time after the environment cue (light) has been introduced to the animal. In these studies, and our own, Zebrafish were raised on 14:10 light-dark cycles, so ZT<14 refers time points with light, while ZT>15 refers to times without light. Here, the ZT is a 24-hour clock (i.e. is in between ZT=0 and ZT=24).
19
expression decreasing markedly after the lights are turned off [119,125]. Due to cry4’s
potential location in cones, expression during times where light information is available, the
inability to participate in circadian regulation, and localization with bound FAD, it appears
to be best suited for LDRPM. In summary, zebrafish appear to have a visual system that could
participate in light-dependent magnetoreception. Is there evidence that these teleosts are
sensitive to magnetic fields?
1.5.2 Magnetoreception behavioral evidence and assays
Magnetoreception in zebrafish has been demonstrated in a variety of ways. One of the
first studies demonstrated that groups of zebrafish were able to be trained to avoid a strong
magnetic field associated with an electrical shock [163]. Since then, bimodal orientation4 has
been demonstrated in adult zebrafish numerous times [40,58,164]. Recently, a unimodal4
(polar) response was shown in adult zebrafish in the dark [40]. Indeed, zebrafish appear to
be sensitive to magnetic fields in light and dark, but the behavioral outputs seem to be
different, suggesting the potential for multiple magnetoreception mechanisms. In agreement
with this, when zebrafish are placed in an extremely strong magnetic field (MRI strength) in
the light, swimming orientations are severely disrupted to the point where they appear to
be flopping out of water, in their tanks [57]. When tested in the dark, this response is not
seen, and instead zebrafish merely increase their swimming speeds and velocities. This
locomotory response was attributed to the vestibular system being overstimulated, but the
affect of light cannot be fully explained by this type of processing. In a somewhat related
4 Bimodal orientation refers to preference along an axis, such as North-South. Unimodal or polar orientation refers to preference to a certain vector such as North or South only.
20
study, larval zebrafish reared in strong magnetic fields appear to have a high proportion of
fused otoliths (usually two distinct inner ear placodes) [56]. In the same study, strong
magnetic fields caused larval fish to orient unimodally parallel in the direction of the
magnetic field. Other behaviors, such as rheotaxis (orientation relative to water flow) has
been shown to be affected by magnetic fields [165]. Specifically, ablation of the lateral line, a
fish specific, mechanosensory organ used to detect vibrations, altered but did not abolish the
magnetic field affect on shoaling zebrafish rheotaxis. These recent studies suggest that
zebrafish may use magnetic fields for a variety of purposes in daily life, whether it is
orienting when shoaling, or navigating from floodplains to streams in their natural habitat
[166,167]. It is likely that the detection of magnetic fields is mediated by multiple systems,
each transducing slightly different magnetic information to the animal. Manipulation of the
lateral line and involvement of vestibular system has been shown in zebrafish
magnetoreception, but other areas such as the eye have yet to be tested.
Despite an increasing number of studies showing that zebrafish have light-specific
magnetoreceptive behaviors, and have the molecular components for LDRPM, few
mechanistic experiments that test the requirement of photoreceptors or Cry exist in fish.
Zebrafish are amenable to various molecular manipulations, including targeted cell ablation,
which is described below.
1.5.3 Nitroreductase-mediated targeted ablation
To ablate specific cell types, transgenic expression of bacterial enzymes can be taken
advantage of. Nitroreductase (NTR) is an enzyme isolated from E. coli that can convert
normally harmless prodrugs such as metronidazole (MTZ) into DNA crosslinking agents,
21
which ultimately causes cell-death via apoptosis [168]. Transgenically, NTR (nfsb gene) can
be expressed under a cell-specific promoter (such as sws1 to ablate UV cones) with no effect
on non-NTR expressing cells [169-171]. In support of this, ablation of zebrafish
photoreceptor subtypes expressing NTR does not create a toxic bystander effect in other
retinal cells [169,170,172-174]. For magnetoreception this is particularly practical since the
location of the magnetoreceptor is unknown. Whole organism gene knockout of Cry would
be useful to determine if Cry is involved but does not point to what sensory system Cry is
specifically interacting with. Additionally, fluorescent proteins fused to NTR have been
created, which allow the visualization of cells before and after ablation [172,174]. If
expressed in a regenerating population, such as the zebrafish cones, regeneration of the cells
can be observed via fluorescent microscopy. For these reasons, cell specific ablation is a
powerful tool that is well-developed in zebrafish. There are many other tools designed to
manipulate zebrafish genetics; those potentially interesting to magnetoreception are
discussed in Chapter 3.
With a growing amount of behavioral evidence, the presence of currently untested
molecular candidates, and the ability to selectively manipulate potential genetic and cellular
mechanisms, zebrafish are ideally suited to study vertebrate light-dependent
magnetoreception.
1.6 Purpose of study/ objectives
The overall aim of this thesis was to generate evidence for a light-dependent,
Cryptochrome-based magnetoreception complex in fish. Using zebrafish, I set out to test
22
whether cry interacted with cone photoreceptors, as seen in migratory birds. Since Cry
activation and magnetoreception seem to largely require short-wavelength light, I focused
on the short-wavelength photoreceptors found in zebrafish (UV and blue). Due to Cry1a’s
association with UV cones in birds, and the potential role that UV cones play in fish
navigation, I hypothesized crys with unknown roles (either cry2 or cry4) would be expressed
in UV cones. To do this, I measured non-circadian cry2 or cry4 mRNA in larval and adult
zebrafish retina using qualitative and quantitative gene expression assays such as in situ
hybridization and RT-qPCR.
The visual system is highly similar between zebrafish and other vertebrates,
including birds, but zebrafish are truly advantageous because they can be used to implement
precise molecular manipulation of retinal photoreceptors. To experimentally test the Cry-
cone association, I utilized targeted cell ablation to selectively ablate UV and blue cones from
larval and adult zebrafish retina to test how cry mRNA expression patterns and levels
changed after cone removal. I predicted that ablation of UV cones would decrease cry mRNA,
while blue cone ablation would not disrupt cry expression. Ablation of neighboring blue
cones served as an important control, to ensure the mere disruption of the cone mosaic (i.e.
any cone being removed) didn’t cause indirect expression changes of cry. I tested Cry-cone
interactions throughout development, to see if any ontogenetic changes were present.
Zebrafish magnetoreceptive behavior has been characterized in adults, and various larval
fish appear to have well-established magnetic compasses but the way in which fish detect
magnetic fields is still largely unknown.
23
Ultimately, the evidence generated here serves to build the molecular basis of fish
LDRPM and will hopefully catalyze further mechanistic investigation into whether UV cones
and/or Cry mediate magnetoreception in fish.
24
Figure 1. The geomagnetic field. The Earth’s magnetic field is similar to a bar magnet, and
somewhat confusingly does not align with our geographic poles. Despite this, we name the
magnetic pole close to the geographical North pole, Magnetic North (mN) and same with the
South pole. The angle between magnetic and geographical poles varies and can be measured
as declination. Intensity is greatest at the poles and weakest at the Equator. Inclination is
shown as green arrows that intersect with the Earth at different angles, with the greatest
angles at the North (red arrow) and South (pink arrow) poles. mN= magnetic North, mS=
magnetic South. Reused with permission from [2].
25
26
Figure 2. Model of light-dependent magnetoreception. A) Cone photoreceptors along the
back of the hemispherical retina serves as an ideal scaffold for magnetoreception. B) Cry
(aqua) may be located in cone outer segments between UV opsin proteins (sws1; magenta)
as seen birds. This relationship allows Cry to be semi-fixed in cellular space and could permit
communication between Cry and opsin, allowing visual transduction to be altered my
magnetic fields. C) After light initiates an electron transfer from a Donor (D) to an Acceptor
(A), both internal and external magnetic fields can alter singlet-triplet interconversion rates
between radical-pairs. In the singlet-state, electrons spin antiparallel in relation to each
other (illustrated by the same orientation of each red arrow). In triplet-state, electrons spin
in parallel, increasing the time spent in signaling form. The time spent in each state could
result in a different signal, such as a neurotransmitter or electrical output, resulting in a
magnetic sensor. Adapted from [2,36].
27
Figure 3. Cryptochrome can create radical-pairs between FAD and Trp. A) Protein
model of Arabidopsis Cry highlighting the three conserved Trp residues in yellow, that
mediate electron transfer between them and FAD co factor. B) Reaction scheme outlining the
creation of the magnetically sensitive radical-pair (RP1) and the protonation of FAD, Trp or
both to create the non-magnetically sensitive radical-pair (RP2). Light excites FAD to the fully
oxidized form sFAD* which is conserved in the singlet-state. Electron transfer creates either
singlet or triplet-state radical-pairs of FAD and Trp depending on the external or internal
magnetic field (red/blue arrows). kC is the equilibrium constant and time it takes for the
radical-pairs to deprotonate (1µs). kS is the time it takes for the singlet RP1 to back transfer
to ground-state. Reused with permission from [33,114].
28
Figure 4. How the magnetic field may alter animals’ visual perception. A)
Magnetoreceptors are shown as red rods and are represented throughout all spatial
orientations of the eye. B) Depending on the orientation of the magnetoreceptor relative to
the magnetic field (black arrows), the resulting output (denoted as black, white or grey
squares) may be different. C) If this interacted with the visual transduction process, it may
cause a change in visual perception across the animal’s visual field. Reused with permission
from [2] based on [36].
29
2 EXPLORING POTENTIAL FOR LIGHT-DEPENDENT MAGNETORECEPTION IN
ZEBRAFISH: CRYPTOCHROME 4 IN A SELECT SUBTYPE OF RETINAL
PHOTORECEPTORS
2.1 Introduction
Many organisms use magnetic fields to orient in local spaces or to navigate long-
distances. Although magnetoreception has been studied extensively, the underlying
mechanisms remain debatable and unknown [175]. Cryptochrome (Cry) is the primary
molecular candidate that creates light-dependent radical-pairs sensitive to magnetic fields
[32,36,176]. Cry is associated with the bird visual system where it is co-localized with both
short- and long-wavelength cone photoreceptors [124,138], and thus is well-positioned for
light-dependant activity. Notably, crys’ location in other animal’s photoreceptors is
unknown. Here, we used zebrafish to determine if fish cry might have a light-dependent
magnetoreceptive role. Zebrafish have six cry paralogs; whereas most of these participate in
circadian clock regulation, the function of cry2 and cry4 remain unknown [118,119]. We
demonstrate that cry4 is expressed in zebrafish retina, principally in the short-wavelength
ultraviolet (UV) cone photoreceptors. Using nitroreductase-mediated cell ablation and RT-
qPCR, we found that cry4 expression decreased when UV cones were ablated, but not when
blue cones were ablated. cry2 expression was unchanged after either UV or blue cone
ablation. Although zebrafish magnetic behavior has only been reported in adults
[40,58,163,164], this work suggests larval fish may also have the molecular framework for
light-dependent magnetoreception. Sockeye salmon fry, larval coral reef fish and larval
30
medaka have been shown to be responsive to magnetic fields [5,40,177], but the mechanisms
remain mysterious. These findings provide one potential explanation, insomuch that UV
cones appear poised for light-dependant magnetoreception via photoreceptor subtype-
specific expression of cry.
2.2 Methods
2.1 Animal Ethics
The Animal Care and Use Committee: BioSciences (an Institutional Animal Care and Use
Committee at the University of Alberta, operating under the Canadian Council on Animal
Care) approved this study under protocol AUP00000077.
2.2 Zebrafish Maintenance
Zebrafish (Danio rerio) were raised according to standard procedures [178]. Larvae were
treated with PTU (1-phenol-2-thiourea) beginning at around 9 hours post fertilization (hpf)
to block formation of melanin pigment [179]. Beginning at 5 days post fertilization (dpf),
larval fish were fed powered fish food. Larvae were maintained in E3 embryo media at 23-
25°C on a 14L:10D cycle until fixation at 8 dpf.
Adult fish were maintained at 28°C under standard fluorescent lights on a 14L:10D cycle and
were fed twice daily with brine shrimp and trout chow [178].
31
2.3 Nitroreductase-Mediated Ablation
Tg(sws1:nfsb-mCherry) or Tg(sws2:nfsb-mCherry) larvae were treated with either 10mM
metronidazole (Sigma-Aldrich, Cat. No. M3761-25G; Oakville, O, MTZ) with 0.1% DMSO in E3
media or 0.1% DMSO in E3 media as a vehicle control for 24 hours beginning at 6dpf. Larvae
were washed three times with E3 media and euthanized at 8dpf at either Zeitgeber Time
(ZT)=4 or ZT=20. Tissues were fixed in 4% paraformaldehyde in 0.1M phosphate buffer +
5% sucrose, pH 7.4 (PFA), overnight at 4°C.
Tg(sws1 or sws2:nfsb-mCherry) adult zebrafish (approximately 1 year old) were treated with
MTZ as described above except that system water replaced E3 media. Fundus imaging
confirmed ablation of cones prior to euthanasia as per published methods [150]. After 6
days, adults were euthanized at either ZT=4 or ZT=20 with an overdose of MS-222. Fish were
cervically dislocated before eyes were removed. Full eye was removed from fish in the day
(ZT=4) or at night under minimal red light (ZT=20) and placed directly into either 1mL of
RNALater (Invitrogen, AM7020) or 4% PFA on ice. Retinas were removed after eye
dissection.
A complete description of fish lines used in this study can be found in Table 1.
2.4 Riboprobe production and Fluorescent In-Situ Hybridization
Riboprobe template primers with a T7 transcriptase binding site
(TAATACGACTCACTATAGGG) were used to make template for riboprobe synthesis against
cry paralogs (Table 2). Template was confirmed with the presence of a single band after gel
32
electrophoresis. Riboprobes to opsins were synthesized from plasmids as previously
described [161]. DIG labelled cry and FLR labelled sws1 riboprobes were made as previously
described [180]. Reverse transcription was mediated by T7 polymerase overnight at 37⁰C.
Riboprobe concentration and quality was determined by Nanodrop spectrophotometer (GE
Healthcare 28 9244-02) and RNA gel electrophoresis.
Double fluorescent in situ hybridization for sws1, cry2 and cry4 was performed as previously
described [161,180]. Tissue was hybridized with 1ug/ml of riboprobe at 58⁰C overnight.
After SSCTw-stringency washes, and incubation of secondary antibody (1:100; α-DIG-POD,
Roche 11207733910 or α-FLR-POD, Roche 11426346910 diluted) overnight at 4⁰C, the
tissue was incubated in 1:100 tyramide conjugated to AlexaFluor 488, 555 or 647 TSA
SuperBoost™ (Thermo Fisher Scientific, B40912, B40913, B40916). After development of
each fluorescent signal (15-18 hours at 4⁰C for larval sws1, cry2, cry4, 10-15 minutes at 25⁰C
for adult tissues) the antibody was deactivated by incubation in 3% H2O2 in PBSTw for 30
minutes at room temperature, and the next antibody was added. After PBSTw washes, larval
eyes were dissected as described above and flat mounted in 70% glycerol for imaging. Cuts
were made on each side of adult retina, flattened, and mounted in 70% glycerol for imaging.
2.5 RNA Isolation
2.5.1 Whole Larvae
RNA was isolated from pooled samples of n=5 whole larval zebrafish (8 dpf) or single adult
eyes raised on a 14L:10D cycle collected at either ZT=4 or ZT=20. Samples were stored in
33
RNAlater (Ambion AM7021) at 4⁰ C until extraction. Total RNA was extracted from pools of
larval zebrafish using the RNAeasy Mini Kit (Qiagen 74104) according to the manufacturer's
instructions. Samples were homogenized in 600 µl of Buffer RLT containing 1% of β-
mercaptoethanol with a rotor stator homogenizer (VMR 47747-370), and put through an on
column DNAse digestion using Qiagen DNAse I. After Buffer RPE washes, RNA was eluted in
32 µl of Nuclease-free H2O (Ambion 4387936).
2.5.2 Adult Eye
RNA from single adult whole eyes was isolated using RNeasy Lipid/Tissue Mini Kit (Qiagen
74804) according to the manufacturer’s instructions. Samples were homogenized in 700 µl
of Qiazol with a rotor stator homogenizer. After phenol:chloroform extraction, DNAse
digestion and Buffer RPE washes, RNA was eluted in 32 µl of Nuclease-free H2O (Ambion
4387936). RNA quantity was determined by a Nanodrop spectrophotometer (GE Healthcare
28 9244-02) and Agilent 2100 Bioanalyzer (Agilent RNA 6000 NanoChip). All samples had
similar rRNA (18s and 28s) profiles and RNA integrity numbers (RIN values) of 8.0 or
greater.
2.6 RT-qPCR
2.6.1 cDNA synthesis
RNA input was standardized to 500ng per cDNA reaction. cDNA was generated using qScript
cDNA Supermix (Quanta Biosciences 95048-025) as per manufacturer’s instructions. Each
reaction consisted of 10 µl, with x µl of RNA (500ng total RNA each reaction), 2 µl of qScript
34
Mastermix, and x µl of Nuclease-free H2O. cDNA was diluted 1:10 for following RT-qPCR
experiments.
2.6.2 RT-qPCR Parameters
Reverse transcription quantitative-PCR (RT-qPCR) experiments follow MIQE guidelines
[181]. RT-qPCR was completed using a 7500 Real-Time PCR system (ABI, Applied
Biosystems) with 2X QPCR Mastermix (*Dynamite*) (MBSU, University of Alberta).
Transcript abundance for cry2, cry4, sws1 and sws2 was assessed relative to β-actin, an
endogenous housekeeping gene optimized for zebrafish [182,183] (and see Endogenous
Control Stability Assay). Technical replicates were performed in triplicate. Biological sample
sizes are stated for each experiment in respective figure legends.
2.6.3 Endogenous Control Stability Assay
To ensure β-actin was a suitable endogenous control, an endogenous stability assay was
performed (Figure 11). Average Ct values were compared between n=3 DMSO and n=3 MTZ
treated pools of larvae after blue and UV cone ablation. Ct values ranged from 17.90 to 19.80
(Average = 19.01±0.39). Each biological replicate was tested in triplicate.
2.6.4 Primer Validation
RT-qPCR primers were designed using Primer Express 3.0 software and verified with a
standard serial dilution to determine efficiency [181]. Primers were designed at the end of
the 3' exon in each gene or the 3' UTR and were expected to amplify all splice variants. Primer
35
sequences and efficiencies are available in Table 3, where a slope of <0.1 indicates the
efficiency of the target amplification is approximately equal to the reference amplification,
and thus the primers are suitable for the experiment [184]. To assess primer specificity
disassociation curves were analyzed, and only primers producing a single peak indicating a
specified product, were used.
2.7 Microscopy and Imaging and Figure Assembly
Fluorescent images were taken on an LSM 700 inverted confocal microscope mounted on a
Zeiss Axio Observer.Z1, and captured using ZEN 2010 software (version 6.0, Carl Zeiss AG,
Oberkochen). 63X oil (NA =1.4) or 40X water (NA 1.4) objectives were used to image flat
mounted larval or adult zebrafish retina. Figures were assembled in PowerPoint (version
1710, Microsoft Office 365 ProPlus). Images were manipulated for contrast & brightness in
Zen 2010. Cartoon zebrafish were obtained under a free culture Creative Commons license
(CC BY-SA 4.0) from Mind the Graph (Version March 03, 2016, Mind the Graph LLC,
www.mindthegraph.com).
2.8 Statistics
For RT-qPCR experiments n>3, Mann-Whitney U Tests were calculated and performed in
GraphPad Prism (Version 7.02 for Windows, GraphPad Software, La Jolla California USA,
www.graphpad.com). For RT-qPCR experiments n=3, unpaired t-tests with Welch’s
correction were performed in GraphPad Prism. Data is presented as mean ± standard error
36
of the mean (SEM). Significance is denoted in figure legends (p at least<0.05). Each pool
(n=5) of larval 8dpf zebrafish and each single adult eye is considered as an independent
biological replicate.
2.3 Results and Discussion
2.3.1 Expression of cry4 in larval zebrafish retina
Cry has been suggested to function in light-dependent magnetoreception in flies,
cockroaches, butterflies and a variety of birds [13,14,71,143] but its role in fish
magnetoreception has largely been overlooked. Many fish undertake long distance
migrations, exhibit magnetic behaviour [5,9,21,24-26,40,58,60,163-165,185] and contain
various crys [117-119,125], but little is known about the cellular location and function of
these putative magnetoreceptors. To test if cry2 or cry4 mRNA was expressed in zebrafish
UV cones (which express sws1 opsin), double fluorescent in situ hybridization was
performed on larval zebrafish aged 8 days post fertilization (dpf) that were fixed at midday
(at zeitgeber time (ZT) = 4). cry2 expression was apparent in the focal plane where cone
photoreceptors reside, but did not strongly overlap with sws1 opsin expression, suggesting
it is not highly expressed in UV cones (Figure 5A-A’’’), but perhaps is in other photoreceptor
subtypes. cry4 showed stronger expression throughout the retina and overlapped
consistently with sws1 expression (Figure 5B-B’’’). cry4 was expressed within the vast
majority of labelled UV cones, and was not detected within neighbouring photoreceptors,
suggesting it is UV cone specific. Expression of cry2 and cry4 has previously been described
in larval zebrafish retina as being broadly distributed throughout all retinal layers at 5dpf,
at least during time points in which they are highly expressed (ZT=23, ZT=15 respectively)
37
[119]. Specifically, cry2 and cry4 have been shown to be expressed strongly in the larval
outer nuclear layer (ONL) where photoreceptors reside [119], in a cyclic circadian manner
[118]. The results presented here support a cellular localization for cry4 in larval zebrafish
UV cones during the day.
2.3.2 cry4 is specifically expressed in the UV cone subtype of larval zebrafish
The implied expression of cry within a specific photoreceptor subtype was very
intriguing, as cry is the best candidate proximate mechanism of light-dependant
magnetoreception. To validate that cry4 was expressed within UV cones, a combination of
pharmacological and transgenic technologies was used to specifically ablate the short-
wavelength cones. Tg(sws1:nfsb-mCherry) and Tg(sws2:nfsb-mCherry) are well-
characterized transgenic lines that have nitroreductase (NTR) expressed within UV or blue
cones, respectively [174]. NTR expression alone appears to be inert to the cells. After
addition of a prodrug (Metronidazole- MTZ), NTR converts MTZ into a cell-autonomous
cytotoxin that causes DNA cross linking and induces the cells to undergo apoptosis
[170,172]. Previous work supports that this technique is cell-specific and has no discernable
bystander effects on adjacent photoreceptors [170,173,186].
As seen in untreated larvae (Figure 5B), we found that control fish with UV cones
(NTR-transgenic larvae treated only with vehicle control DMSO) expressed cry4 within UV
cones (Figure 6A’-A’’’). Following ablation of UV cones by treating NTR-transgenic larvae
with prodrug MTZ, cry4 abundance was dramatically decreased throughout the larval retina
(Figure 6B’-B’’’) and was apparent only within the few surviving UV cones. RT-qPCR
38
confirmed this, as cry4 mRNA levels decreased approximately 60% after UV cone ablation
(Figure 6C; Mann-Whitney U, p=0.007). On the other hand, cry4 did not significantly change
in abundance when the neighbouring blue cones were ablated (Figure 6E p>0.9999). cry4
expression was also unchanged when non-transgenic control larvae (AB/Wik strain) were
treated with MTZ, confirming that the addition of the prodrug MTZ does not itself
measurably influence cry4 expression (Figure 6E, p= 0.7182). Further, a paralogous gene
cry2, was not found to have its transcript abundance altered by ablation of UV cones
(p=0.1081) or blue cones (p>0.9999) (Figure 9), suggesting a specialized expression pattern
for cry4.
The localization of cry4 to UV cones in larval zebrafish was thoroughly validated,
because two independent methods confirmed that cry4 abundance substantially decreased
when UV cones were ablated; this did not occur when blue cones were ablated, and a
paralogous gene, cry2, was unaltered in its abundance when UV cones or blue cones were
ablated.
These findings suggest larval zebrafish retina contains the molecular basis of a light-
dependent magnetoreception complex. Furthermore, cry4 appears to be well-positioned, i.e.
localized within a subtype of retinal cone photoreceptors, to mediate light-dependant
mechanism(s). Although currently there is sparse evidence for larval zebrafish being
magnetoreceptive [40,56], larval coral reef fish have a well-established magnetic compass
that is primarily used when celestial compasses (sun/star) are unavailable [24,177]. cry4’s
localization in zebrafish UV cones at ZT=4 (midday in our rearing conditions, 1200 MDT)
coincides with a time when the sun is stereotypically directly overhead a tropical animal and
39
may not provide useful directional information. Indeed, at these time points, larval coral reef
fish have near-random orientation when tested for sun compass orientation behavior [187].
Although this time varies seasonally and geographically (i.e. the sun is not always directly
overhead at ZT=4 or 1200 MDT), and polarized light may influence magnetic orientations at
solar noon [75], this could be a general time point where light-dependent magnetoreception
would be useful. It would be interesting to see cry4’s expression in the retina of larval reef
fish who have well-defined magnetic compasses.
2.3.2 cry4 is expressed in adult zebrafish UV cones
Next we tested if cry4 remains associated with UV cone photoreceptors throughout
ontogeny into adulthood, where zebrafish magnetoreceptive behaviour has been
demonstrated [40,58,163,164]. In adult zebrafish, cry4 was found to be abundantly
expressed in UV cones (Figure 7A-A’’). As above, a paradigm ablating UV cones in adult fish
at midday (ZT=4) demonstrated a concerted decrease in cry4 abundance (Figure 7B-B’’).
Quantitatively, cry4 mRNA was decreased approximately 40% at ZT=4 following UV cone
ablation (Figure 7C, ZT=4, p=0.041; Figure 7B).
In the adult zebrafish retina, cry4 has previously been shown to be expressed in the
ONL and the inner nuclear layer (INL) at ZT=4. In the ONL at ZT=4, it was suggested that cry4
might be localized to cone photoreceptors [119]; that interpretation lends further support
that UV cones are the specific photoreceptors in which cry4 is expressed in. cry4’s abundance
is also know to vary over circadian time [119,125]; thus, we considered if cry4 might have
different abundances at night following cone ablation. At night (ZT=20; 0400 MDT), UV cone
40
ablation did not measurably change cry4 expression (Figure 7E; p=0.09). This was expected,
as cry4 expression appears to be almost non-existent in the zebrafish retina at this time point
[119]. The remaining cry4 transcript that was detected is likely in other retinal cell types
(not UV cones). The significant decrease of cry4 between ZT=4 and ZT=20 (Figure 7E;
p=0.031) reinforces that cry4 abundance appears to be cyclic in the retina [118,119,125]. In
other animals, such as zebra finch, European robin and chicken, cry4’s expression in the
retina appears to be constant throughout circadian time [124,188], irrespective of the
presence of light. The effect of light on turnover rates of Cry4 protein is currently unknown,
but when birds are exposed to 30 minutes of complete darkness, activated Cry1a that is
usually localized in bird UV cones is not detectable in the retina [189].
2.3.3 Blue cone ablation does not measurably disrupt cry4 abundance in adult retina
Ablation of blue cones in adult zebrafish at either ZT=4 or ZT=20 (Figure 8A-A’) did
not change cry4 mRNA levels (Figure 8B; ZT=4 p=0.771; ZT=20 p=0.856) suggesting that
cry4 is not expressed in adult blue cones. A significant decrease of cry4 from ZT=4 to ZT=20
was observable irrespective of blue cone ablation (Figure 8B; DMSO p=0.048; MTZ p=0.011)
as described elsewhere (Figure 8, [119]). This data supports that cry4 expression in the
short-wavelength cones is primarily restricted to the UV cones in both larval and adult
zebrafish. The apparent absence of cry4 in blue cones, and its abundance in UV cones, could
suggest a cellular connection with magnetoreceptive purpose; it has been theorized that to
separate visual information from magnetic information, two side-by-side receptors that
receive the same light input would be beneficial [33,124]. The most immediate retinal cells
that compare information between adjacent photoreceptors are the horizontal cells in the
41
INL. Outputs from UV and blue cones are processed by a specific type of horizontal cell (H3
cells) [190-192]. The separation of magnetic signals could be accomplished by the output of
cry4-containing UV cones being compared to the output of neighbouring, similarly short-
wavelength-sensitive, non-cry4 containing blue cones by H3 cells [139]. Additionally,
zebrafish UV and blue cones were recently described to be connected at the synaptic level
via telodendria [186].The spectral sensitivities of UV and blue cones overlaps significantly at
about 375 nm-400 nm in zebrafish [156,193,194] and the interesting synaptic connections
they make at the cone pedicle suggests they are sharing information at the receptor level
before sending signals downstream for further processing. This type of “immediate” signal
integration could point to the retina’s early efforts to disentangle magnetic and visual
information.
2.3.4 cry4 and cone photoreceptors as potential magnetoreceptors in fish
In summary, our results demonstrate that UV cones in larval and adult zebrafish
express a putative molecular magnetoreceptor, cry4. This was observed in midday time
points (ZT=4) and was disrupted by UV cone ablation but unaltered after blue cone ablation.
Additionally, paralogous cry2 was unchanged after UV and blue cone ablation in larval and
adult zebrafish. Although it is still unknown if cry4 or any Cry functions as a magnetoreceptor
in vertebrates, it appears the relationship between Cry and cone photoreceptors is present
in a diverse range of taxa. In European robins and chicken, Cry1a is localized in the UV cones
and is active when exposed to short-wavelengths that permit magnetic orientation behavior
[68,189,195]. Cry1 is also found in S1 short-wavelength cones (‘blue cones’ homologous to
UV cones of birds and fish) in a variety of mammalian species [140]. cry4’s retinal location in
42
other fish has yet to be described, but in birds, Cry4 has recently been shown to clearly be
localized in the long-wavelength single cones and double cones of European robin and
chicken [124]. Why the cellular location of Cry4 may be different between fish and birds is
currently unknown; the very divergent life histories of zebrafish and migratory birds cannot
be ignored. The differences in the spectrum of available light in air versus water [196-198]
could also point to why different photoreceptors may contain cry4. Additionally,
photoreceptor subtypes vary significantly in birds when compared to zebrafish; birds have
individual red and green cones and unique paired double cones that express a specific opsin
not found in fish [199-202], while zebrafish have paired double cones that have red and
green sensitive counterparts [156,161,203]. cry4 may also be lowly expressed in the
red/green double cones of zebrafish, but this requires further investigation. In support of UV
cones being important for fish navigation, they have previously been suggested to mediate
the detection of polarized light [204,205]. Also, when migratory salmon reach sexual
maturity and return to their natal streams, a population of UV cones regenerate after most
degenerate during metamorphosis [206-208]. It is tempting to suggest a magnetoreceptive
link with cry4’s expression in UV cones, but more work is needed to formally test this idea.
These among other recent findings provide exciting evidence that fish may contain a
light-dependent magnetoreceptor. New, robust behavioral experiments [26,40] utilization
of genome editing (CRISPR-Cas9; [209]) next gen RNA-Sequencing after magnetic field
manipulations [210,211] and advances in visualizing neuronal activity (Ca2+ imaging;
[212,213]) will be key in determining the requirement of Cry and cone photoreceptors in
fish magnetoreception.
43
Table 1. Transgenic zebrafish lines used in experiments.
Table 2. Primers used for Cryptochrome riboprobe synthesis.
Genotype Description ZFin ID Reference
Tg(sws1:nfsb-
mCherry)
This line expresses nitroreductase (nfsb
gene)-mCherry fusion protein in UV
cones (sws1). Nitroreductase converts
prodrugs such as MTZ into DNA cross-
linking agents, inducing targeted
ablation via apoptosis. The mCherry tag
allows fluorescent visualization of these
cells.
ZDB-ALT-
080227-1
[174]
Tg(sws2:nfsb-
mCherry)
This line expresses nitroreductase (nfsb
gene)-mCherry fusion protein in blue
cones (sws2), which can allow for
targeted ablation as described above.
ZDB-ALT-
160425-3
[174]
Gene ZFin ID Primer sequences (forward, reverse; T7 promoter sequence
in red)
cry2
ZDB-
GENE-
010426-6
F: 5’ CCCTTGTCGCTCTTTGGTCA ‘3
R: 5’ TAATACGACTCACTATAGGGACTGCCGTTCCTGTTTTCCT ‘3
cry4
ZDB-
GENE-
010426-7
F:5’ GACAGTGGCGCAGGAAAATG ‘3
R: 5’TAATACGACTCACTATAGGGCGCACCTGACGCATCAATTC ‘3
44
Table 3. RT-qPCR primers used for Cryptochrome mRNA quantification.
† β-actin transcript abundance was found to be stable across treatments herein; Ct values
not significantly different when UV or blue cones were ablated (Also see Figure 11).
Gene ZFin ID Primer sequences
(forward, reverse)
Amplicon
length (bp)
Slope of ΔCT vs. log
(input)
Amplicon
location
(exon #/
total exons)
cry2
ZDB-
GENE-
010426
-6
F: 5'-
GCAGTGACCGCAGGT
TGAA-3'
R: 5'-
GGATGGTTCGGAGAG
GTGAA-3'
78 0.02 3’UTR (11
exons)
cry4
ZDB-
GENE-
010426
-7
F: 5'-
GGCTGCATCATCGGT
AAAGAC-3'
R: 5'-
CGCATCAATTCCAGGT
TCCT-3'
80
0.09 9/11
sws1
ZDB-
GENE-
991109
-25
F: 5'-
TCCTCCCGCAGCACAT
TTAC-3'
R: 5'-
AAAGTTACGGGATTT
GAACAATCAG-3'
80 0.07 5/5
sws2
ZDB-
GENE-
990604
-40
F: 5'-
CTATCTTTGCAATCTG
GGTGGTT-3'
R: 5'-
AAAGGCAGGAGGGAA
TGGTT-3'
78 0.09 4/5
β-
actin
ZDB-
GENE-
000329
-1
F: 5'-
CGGACAGGTCATCACC
ATTG-3'
R: 5'-
GATGTCGACGTCACAC
TTCA-3'
136 Validated in [183] †
4-5/5
45
Figure 5. Larval zebrafish UV cones express cry4 during the day. A-A’’’) Double
fluorescent in situ hybridization on flat-mounted larval zebrafish retinae reveals cry2 mRNA
(magenta) is not co-expressed with sws1 opsin (green), a marker for UV cones. B-B’’’)
Riboprobe against cry4 mRNA reveals overlapping expression with sws1, suggesting they are
co-expressed. Tissue was collected at 8 days post fertilization (dpf) 4 hours after lights were
turned on (Zeitgeber Time, ZT=4). Tg(sws1:nfsb-mCherry)= UV cone ablation line, where UV
cones can be ablated upon the addition of a prodrug MTZ. Larvae were not treated in this
experiment. Scale bars = 20 μM for both magnifications. White square denotes Inset location.
Inset = 10x magnification.
46
47
Figure 6. Ablation of UV cones, but not blue cones, drastically decreases cry4 in larval
zebrafish. A-A’’’) DMSO vehicle control treated larval retina shows cry4 (magenta) is co-
expressed with UV cones (sws1; green). B-B’’’) Tg(sws1:nfsb-mCherry) retinae treated with
prodrug metronidazole (MTZ) to induce ablation of UV cones shows decreased sws1 and cry4
expression. The remaining UV cones continue to express cry4. C-D) mRNA levels of cry4 and
sws1 (respectively) were reduced after treatment and MTZ (red) when compared to DMSO
(grey) as measured using reverse transcription quantitative-PCR (RT-qPCR). mRNA
abundance was normalized to levels of the endogenous control β-actin and standardized to
vehicle control (DMSO) fish. Abundances are plotted as mean with SEM. E) cry4 is not
decreased after blue cone ablation in larval zebrafish Tg(sws2:nfsb-mCherry), as quantified
by RT-qPCR. AB/Wik serves as a non-transgenic control, showing addition of the prodrug
and vehicle control does not alter endogenous cry4 mRNA levels. F) sws2 opsin (blue cone
opsin) is decreased after addition of MTZ, ensuring ablation and RT-qPCR are sufficient
experimental methods. G) Summary of treatments on zebrafish lines. Tissue was collected
at 8 days post fertilization (dpf) 4 hours after lights ON (ZT=4); n= number of biological
replicates (one biological replicate = pool of 5 individual 8dpf fish); Scale bars = 20 μM;
Tg(sws1:nfsb-mCherry) = UV cone ablation line, Tg(sws2:nfsb-mCherry) =blue cone ablation
line; DMSO=Dimethyl sulfoxide. Statistical tests: C-D) Mann-Whitney U Test, E-F) Unpaired
t-test with Welch’s correction; *=p<0.05, **=p<0.01; SEM= Standard Error of the Mean.
48
Figure 7. UV cone ablation decreases cry4 expression is adult zebrafish retina. A-A’)
Live fundus imaging reveals MTZ is sufficient to ablate UV cones in adult Tg(sws1:nfsb-
mCherry) retina. mCherry is fused to nitroreductase (nfsb) and expressed in UV cones.
Zebrafish were treated with MTZ or DMSO for 24 hours and allowed to recover for 5
additional days. After 6 days post treatment, eyes were collected at either 4 -hours after lights
were turned on (ZT=4) or 6 hours after lights were turned off (ZT=20) in the dark under red
light. RT-qPCR was performed as described in STAR Methods. B-C)’’ cry4 (magenta) is
expressed in UV cones (green) and decreased after UV cone ablation as seen in larvae at
ZT=4. D) sws1 opsin is significantly decreased after UV cone ablation at both ZT=4 and ZT=20
which causes E) a significant decrease in cry4 after UV cone ablation at ZT=4, but not ZT=20.
cry4 is significantly decreased from ZT=4 to ZT=20. Scale bars: A-A) = 50 µM, B-C’’) = 20 µM,
n= single whole fish eye from different individuals. Statistical test: Mann-Whitney U Test,
*=p<0.05, **=p<0.01, ***=p<0.001; SEM= Standard Error of the Mean.
49
Figure 8. Effective blue cone ablation does not disrupt cry4 expression in adult
zebrafish retina. A-A’) Live fundus imaging shows blue cone ablation was effective after
MTZ treatment on Tg(sws2:nfsb-mCherry adult zebrafish for 24 hours. mCherry marks
nitroreductase (nfsb) in blue cones. B) cry4 expression was unchanged after blue cone
ablation at ZT=4 and ZT=20 but was significantly decreased from ZT=4 to ZT=20. C) sws2
(blue opsin) is decreased after MTZ treatment at both ZT=4 and ZT=20. Scale bars: A-A) = 20
µM, B-C’’) = 20 µM, n= single whole fish eye from different individuals. Statistical test: Mann-
Whitney U Test, *=p<0.05, **=p<0.01; SEM= Standard Error of the Mean.
50
Figure 9. cry2 expression is not changed significantly after both UV cone and blue cone
ablation in larval and adult zebrafish. After UV cone ablation A-B) cry2 mRNA is
unchanged in 8dpf larval zebrafish at ZT=4 and in adult eye at ZT=4 and ZT=20. C-D) Blue
cone ablation does not change cry2 in 8dpf larval zebrafish at ZT=4 and in adult eye at ZT=4
and ZT=20. For adult RT=qPCR: n= single whole fish eye from different individuals. For larval
RT-qPCR: n= number of biological replicates (one biological replicate = 5 individual 8dpf fish.
Statistical tests: A-C) Mann-Whitney U Test, B-D) Unpaired t-test with Welch’s Correction,
SEM= Standard Error of the Mean.
51
Figure 10. No cry4 probe control for double fluorescent in situ hybridization on adult
retina. In situ hybridization was performed as described (See STAR Methods). In the
magenta channel, no signal is seen when no cry4 probe is added. Alexa488 tyramide (pseudo-
colored magenta) was added, and colour reactions were developed as described. DIC=
Differential interference contrast; Scale bar= 20 µM.
52
Figure 11. β -actin is a suitable endogenous control for RT-qPCR experiments. Squares
denote biological replicates (n=5 whole larvae) treated with either DMSO or MTZ. Tissue was
collected at 8dpf at ZT=4. Ct= Cycle threshold; SD= Standard deviation. Primer sequences are
found in Table S3.
53
3 GENERAL DISCUSSION AND FUTURE DIRECTIONS
This work provides evidence that larval and adult zebrafish UV cones express a
putative light-dependent magnetoreceptor, cry4. How (and if) cry4 and cone photoreceptors
process magnetic information remains unknown, but in a wide variety of taxa, cry appears
to be associated with these cells. Short-wavelength photoreceptors such as UV cones appear
to be particularly suited for magnetoreception due to the localization of cry in their outer
segments and their spectral sensitivity, which overlaps with magnetoreceptive behavioral
outputs [73,124,138]. The retina seems poised to receive magnetic signals based its
hemispherical shape and the orientation of cone photoreceptors. One notable feature of the
adult zebrafish retina is its mosaic nature, where rows of alternating UV and blue cones are
separated by rows of green and red double cones [160,161,193]. Other teleosts such as
flounders, trout, goldfish, guppies and medaka also have highly-ordered photoreceptor
mosaics [157,158,214-218] that vary from the zebrafish arrangement. The function of these
highly ordered cellular networks remains mysterious and a potential role in
magnetoreception is discussed in section 3.1 below.
3.1 The role of UV cones and cone mosaics in magnetoreception
To separate visual information from magnetic information it has been suggested that
a system with two side-by-side photoreceptors, that receive the same light input, would be
beneficial [33,124]. In this system, magnetic signals can be filtered from light signals by
comparison between both photoreceptors. Horizontal cells (HCs) are well-characterized
retinal neurons that receive input from cone subtypes. HCs also send feedback signals to
specific photoreceptors, which helps improve contrast and color constancy [190,191,219].
54
In zebrafish, H3 cells are specifically innervated by UV and blue cones, suggesting
information is being compared between these two photoreceptors [191,192]. The
abundance of cry4 in UV cones and apparent absence in neighboring blue cones (Figure 5-9)
could suggest a cellular connection with magnetoreceptive purpose. The spectral sensitives
of UV and blue cones overlaps significantly at about 375 nm-400 nm in zebrafish
[156,193,194], allowing for a similar light stimulus to be processed between these two
photoreceptors. The separation of magnetic signals could be accomplished by cry4-
containing UV cones comparing their output to neighboring, similarly short-wavelength
sensitive, non-cry4 containing blue cones via H3 cells. This could theoretically allow the
retina to compare light + magnetic information (via UV cones) to light only information (via
Blue cones), allowing the magnetic signal to be filtered out. Additionally, zebrafish UV cones
and blue cones are connected at the cone synaptic level via fine processes called telodendria
[186] suggesting they are communicating prior to sending signals to HCs. Telodendria have
been proposed to add visual acuity and decrease signal to noise ratio, which could be useful
if magnetic signals alter visual perception [186,220,221]. This type of immediate signal
integration could point to the retina’s early efforts to disentangle magnetic and visual
information. This system has been proposed to function in European robins which harbor
Cry1a in their UV cones [138,139]. In this case, robins exhibit magnetoreceptive behaviors
when exposed to short-wavelength light that encompasses UV cone and blue cone activation
[69], which coincides with activated Cry1a detection in the retina [189].
Could UV cones and cry4 be interacting similarly in the zebrafish retina? The
maximum wavelength sensitivity (λmax) of zebrafish UV cones (~360 nm; [156]) is
remarkedly similar to the absorption spectra of the fully oxidized version of FAD (~370 nm
55
for SFAD* or FADox in Figure 12) and the magnetically sensitive radical SFAD•− or TFAD•−
(~370 nm) of Cry [14,222-225]. The matched spectra of UV opsin and FAD in redox states
that are required for Cry activation, and creation of magnetically sensitive radicals, points to
UV cones potentially being co-opted for magnetoreception. Interestingly, blue cones λmax
(~410 nm) is still encompassed within the absorption spectra of both SFAD* and FAD•− but
falls on a sharp decrease and plateau of FAD•− ‘s absorption spectra (Figure 12). Co-activation
of blue cones and Cry from the same wavelength is less likely when compared to UV cones:
this further supports that blue cones are not as useful for direct magnetic detection and may
be functioning to help UV cones filter light and magnetic information. Although the cellular
architecture of UV cones appears to make them ideal for LDRPM, are the visual outputs from
these photoreceptors useful for magnetoreception?
3.1.1 UV vision may be suited for visually perceiving magnetic fields
Evidence for UV vision is well-established in a variety of animals, including fish (for
recent review see [226]). Prey capture, mate selection and polarized light detection are some
of the proposed functions of UV vision [227]. Although UV light has been suggested to be
processed as a colour in fish [228,229], how these wavelengths are perceived remains
unknown. Recently, an in-depth study outlined potential ecological implications for
zebrafish UV monochromatic vision [197]. Neural circuits for UV vision appear to be most
suited to detect stimuli in the upper and frontal visual field (i.e. directly above the animal).
At the photoreceptor level this coincides with a dense population of UV cones in the larval
zebrafish ventral-temporal retina [197]. In this area of the visual field, it is thought that little
56
colour information is processed, and instead visual input is predominated by silhouettes/
shadows created by UV reflection and penetrance. Indeed, UV vision as a basis for prey
detection has been suggested in zebrafish [197,230,231], as common food items such as
unicellular paramecia scatter light that is biased to UV [167,232]. A visual circuit designed to
detect short-wavelength shadows and silhouettes could be useful if it was also processing
magnetic signals. It has been argued that a cone-mediated magnetoreceptor would be non-
functional during the day because opsins would saturate photoreceptor signals upon photon
absorption [33]. However, in shallow water (where zebrafish are found), short-wavelengths
from sky illumination are mostly lost, shifting the spectrum of available light towards longer
wavelengths [196,197,233]. The absorption peak of zebrafish UV opsin is at the extreme far
end of the short-wavelength light available in the water [196], making these cones unlikely
to function effectively in scenarios that require high signal-to-noise ratio, such as high acuity
colour vision. In support of this, chromatic aberration of short-wavelength light makes the
receptors especially likely to blur a formed image on the retina [226]. As such, UV cones may
be acting to provide a “fuzzy” contrast for zebrafish. This would theoretically be apt for
visually altered magnetosensing, as the receptor thought to mediate this should be contrast-
sensitive, rather than involved in colour input (Personal communication, Dr. John Phillips).
Long-wavelengths that dominate in the water column would be largely used to mediate high
acuity functions while short-wavelengths would be used for contrast detection. The idea that
short-wavelength photoreceptors mediate contrast has been suggested elsewhere
[227,234]; blue photoreceptors have been proposed to constantly adjust a fish’s sensitivity
to the environmental background [216]. For these reasons, it appears UV cones and UV
vision are well-suited to participate in magnetoreception within aquatic environments.
57
3.1.2 Fine tuning of visually mediated magnetoreception may be accomplished by
long-wavelength photoreceptors
Despite a large body of evidence suggesting short-wavelength photoreceptors are
equipped to be magnetoreceptors, the complete story is most likely more complicated. Cry4
was recently found to be localized in photoreceptors sensitive to longer wavelengths [124].
These include chicken and European robin double cones (λmax= ~580 nm) and single long-
wavelength cones (λmax= ~610 nm) [235-237]. Most importantly, Cry4 is clearly not
localized in bird UV cones [124]. This finding is especially interesting when compared to
cry4’s localization to UV cones in zebrafish (Figure 5-7). There are many reasons why this
difference may exist between fish and birds (described in Chapter 2); regardless, it provides
an important discussion point for the potential involvement of long-wavelength cones in
magnetoreception. Why would Cry4, a short-wavelength sensitive photoreceptive molecule,
be found in long-wavelength sensitive cones? This could be beneficial when trying to
separate cone-mediated visual output and Cry-mediated magnetic output. Since the spectral
sensitivity of red cones and the magnetically sensitive radical of Cry are on the opposite side
of the wavelength spectrum, activation of each would require different portions of the
available light. This would prevent competition for similar wavelengths between opsin and
Cry and ensure each output (magnetic versus visual) was using a distinct portion of light.
Double cones also fulfill the requirement of a similar light stimulus being presented between
photoreceptors, as was discussed with UV and blue cones.
It is possible that cry4 is also expressed in a subset of double cones within the
zebrafish retina, but this requires further investigation. It is worth noting that the many
opsins that define green and red cones have extremely unique expression patterns
58
throughout different developmental time points, and areas within the zebrafish retina [203].
Specifically, expression of lws1 and rh2-4 is restricted to a subset of cones in the ventral adult
retina [203,238,239]. Double cones here would be most sensitive to light directly above and
in front of the animal, similar to where UV cone circuits are proposed to be most active. It
would be interesting to see if cry4’s expression differed spatially throughout the retina and
localized to double cones in ventral areas. Characterizing this type of spatial patterning has
yet to be done for crys in adult zebrafish but in the larval eye, cry4 appears to be expressed
broadly throughout most areas of the retina (Figure 5). The expression of cry4 may tighten
to a spatially restricted subset of photoreceptors as the mosaic is established throughout
development, but this requires further investigation.
Additionally, SFAD*/FADox has a second absorption peak around 440 nm to 480 nm
and FAD•− has one around 480-500 nm, which matches the λmax of the green opsins (rh2-1
to rh2-4; ~480 nm to 505 nm) [156,222,223,225,240]. Since this is within the active
spectrum of Cry, the potentially pro-magneto green cones could add acuity to the general
signal created by UV cones. Indeed, a small number of bipolar cell connections in the
zebrafish retina are UV-green cone specific [241], suggesting they are also sharing
information in the early stages of retinal processing. The green cone signal could add sharp
bright lines to the relatively fuzzy image created by UV cones alone (Figure 4). cry4 in the
longer-wavelength red cones may also add to the acuity of magnetoreception; the absorption
spectra for FADH•, the non-magnetically sensitive radical, is broad, but extends into
wavelengths far above 500 nm [225]. Red cone opsin’s (lws1) λmax is ~565 nm, and only
FADH• of Cry can absorb in this spectral range [156,240]. If cry4 was located in zebrafish red
cones as seen in birds, it could represent the long-wavelength extreme end of cry-cone
59
mediated magnetoreception. Ultimately the signals created by absorption of FADH• would
serve as an “end-point” to a certain magnetic stimulus, where the magnetically sensitive
radical is abolished, and Cry returns to its ground-state (Figure 3). This could theoretically
be visualized as sharp black lines, which complement the overall bright pattern generated
from UV cones and activated Cry, and sharp bright lines generated by green cones and Cry
(Figure 4). The interplay between different photoreceptors is likely if LDRPM results in
altering a fish’s visual perception but needs further exploration before it can be tested.
3.2 cry2 and cry4’s function in zebrafish retina: magnetoreception, circadian
photoreception or neither
The unknown function of zebrafish cry2 and cry4, and the supported role of Cry in
magnetoreception in other models makes them most likely to have a magnetoreceptive role
in zebrafish. However, cry also has known roles in tuning circadian rhythms. crys normally
regulate the circadian clock by entering the nucleus and inhibiting CLOCK:BMAL1 mediated
transcription in an autoregulatory negative feedback loop (for review see [242,243].
Zebrafish cry4 does not contain a NLS and cannot repress activation of the CLOCK:BMAL1
complex [118,125], making it unlikely to participate in core circadian regulation. Another
unique characteristic of zebrafish Cry4 is that it has been isolated with bound FAD [126] and
can undergo light-dependent conformational changes [244,245], which are two
requirements for magnetically sensitive radical-pairs to be created. For these reasons, and
its association with UV cones, cry4 is a promising candidate for light-dependent
magnetoreception in zebrafish. Through similar reasoning, it is possible that cry4 could have
60
a yet to be discovered role in circadian rhythms, as it may be acting as an upstream light-
sensor used to drive gene expression necessary to entrain the circadian clock [243,246,247].
Indeed, cry4 appears to be more evolutionarily related to Type I Crys found in invertebrates,
which provide light-input to the circadian clock [112,119]. It is possible that cry4’s
localization in UV cones at ZT=4 (Figure 5-7) is a consequence of a cell-dependent circadian
rhythm of cry. Expression of cry4 appears to move throughout the retina during the circadian
day: spatial expression starts in the ONL and INL in early hours after lights have been turned
on, and progressively moves forward through the retina into the GCL later in the day [119].
At night, cry4 is almost non-existent in the retina, suggesting the presence of light influences
this cycle [119,125]. It has been suggested that cry4 cycles in a similar pattern in constant
light or dark conditions, but these experiments need to be validated [125]. For now, it is
unknown if zebrafish cry4’s expression is regulated exogenously (light-dependent) or it
cycles endogenously. Regardless, an alternative role for cry4 in circadian input is possible.
Further experiments will be required to elucidate cry4’s role in UV cones, and in zebrafish in
general.
cry2’s role in zebrafish biology is even more mysterious. cry2 is found in both the
nucleus and cytoplasm but has lost it’s NLS. Additionally, it can only weakly bind CLOCK:
BMAL1, and is predicted to be insufficient in regulation of circadian transcription [118]. In
larval retina expression of cry2 did not overlap with UV opsin, suggesting cry2 is not localized
to UV cones in larval stages (Figure 5). Despite this, although not statistically significant, cry2
expression did decrease close to the magnitude of cry4 after UV cone ablation at ZT=4 in
adult zebrafish (Figure 9). cry2 may be very lowly expressed in adult UV cones, or its
expression may somehow be regulated by the presence of UV cones, but this requires further
61
exploration. It was previously suggested that cry2 is expressed in zebrafish short-
wavelength photoreceptors at ZT=23 [119], but effective ablation of either UV or blue cones
did not decrease cry2 in the adult retina at ZT=20 (Figure 9). Recently, transcriptional
profiles of zebrafish rods suggest that cry2 is highly expressed in these photoreceptors
compared to other retinal cells [248]. Cry-rhodopsin interactions have been speculated in
the magnetoreception literature [249,250], but rods extreme sensitivity to low levels of light
makes them unlikely to function as a daytime magnetoreceptor [139]. It is currently
unknown if cry2 has the appropriate biochemical underpinnings to create magnetically
sensitive radical-pairs, but theoretically if it does, cry-rod interactions may mediate
magnetoreception in scotopic scenarios.
The role of cry and cone photoreceptors in fish magnetoreception can only be inferred
from molecular association. To test functional significance: behavioral assays, genetic
manipulation and neuronal recordings must also be performed.
62
3.3 Future Experiments to test LDRPM in Zebrafish
3.3.1 Behavioral experiments to test for LDRPM
Evidence for magnetoreception has been built off behavioral observations in a
staggering number of animals. To properly test the role of UV cones and cry in
magnetoreception, behavioral experiments after manipulation of these variables must be
performed. Without an innate compass preference or a migratory heading, testing
magnetoreception mechanisms is less intuitive in zebrafish when compared to migratory
birds or fish. The first step in doing so, is to define a robust, repeatable magnetic behavior.
Bimodal orientation (the preference to align along an axis) has been demonstrated a number
of times in zebrafish, and is observed when fish have access to light and would theoretically
be using LDRPM [40,58,164]. However, this behavior appears to be innate, and is not
constant across zebrafish (i.e. individual fish don’t all have the same bimodal orientation)
making it a potentially difficult behavior to evaluate after manipulation of UV cones or cry.
An alternative experiment may include motivating the animal to respond to a
magnetic stimulus. This could be done by giving zebrafish a reward (food) that is paired to a
magnetic vector (such as North). One could use a 4-arm plus maze and manipulate the
magnetic field, so each arm would represent a magnetic direction. Through conditioning
experiments, removal of food should cause zebrafish to spend more time in the arm of the
maze that is associated with the trained magnetic vector (North). A problem with these
experiments is that the motivation of the zebrafish can result in poor response rates.
Limiting food intake before experiments would ensure that zebrafish were primed to search
63
for food, but if zebrafish were tested in a relatively inconsequential environment, they may
search for food randomly in each arm and ignore magnetic cues. To add extra motivation,
the arms of the maze could be inclined, causing lower water levels further into the arm of the
maze (a risky environment for zebrafish; Personal communication, Dr. John Phillips). If food
was only given in these shallow sections, and was paired with the magnetic stimulus,
zebrafish may expend more energy on choosing the right arm via magnetic cues, in attempt
to spend less time in a risky environment. Additionally, increasing the potency of the reward
may effectively reveal magnetic behaviors. Recently, zebrafish were rapidly trained to self-
administer opioids by swimming over a sensor [251]. This assay also worked with food
rewards, but fish took much longer to show learned responses. Using drugs as a reward has
many downfalls but this work could at least be used to promote research into characterizing
non-harmful substances that are effective in zebrafish training assays. Regardless of assay,
LDRPM has rarely, if ever, been tested in zebrafish, leaving many critical experiments open
to complete.
An intuitive next step would be to test if magnetically induced training to a reward
was wavelength specific. If LDRPM via cry was responsible for this behavior it would be
predicted that training would be possible in monochromatic short-wavelength light (that
encompassed UV cone and FAD activation) but would be inhibited in monochromatic long-
wavelength light (outside activation spectra of UV cones and FAD). Additionally, this
behavior should be limited if zebrafish were tested in complete darkness. Despite this logic,
zebrafish have been shown to exhibit magnetic behaviors in the dark [40,56], making it very
likely that an alternative magnetoreception mechanism exists. To tease apart these
mechanisms, targeted cone ablation could be used to test if cry4 expressing UV cones were
64
required for magnetic behaviors. If zebrafish were using LDRPM, trials in short-wavelengths
should be influenced by UV cones/ cry. After ablation of UV cones, it would be predicted that
the behavioral response would be diminished. If another LDRPM-independent mechanism
was compensating, a complete abolishment of the trained response may not be observed,
and instead, only a moderate decrease may be seen after UV cone ablation in short-
wavelengths. Additionally, if a trained response was observed in darkness or long-
wavelengths (in favour of another mechanism) it would be predicted that UV cone ablation
would not substantially affect the behavior in these trials.
Use of monochromatic wavelengths and ablation of UV cones could also be used to
test innate bimodal orientation. Here, it would be also predicted that the behavior would be
observed in short-wavelengths sufficient for UV cone and cry activation and decreased/
unseen after UV cone ablation, or during exposure to monochromatic long-wavelength light.
Bimodal orientation has only been demonstrated during trials when zebrafish are exposed
to light, making it a promising behavior mediated by LDRPM. In the dark, zebrafish exhibit a
unimodal response (align towards one vector only, such as North), which may be mediated
by magnetite or some other yet to be found receptor.
As discussed in Chapter 1, other diagnostic tests for LDRPM also exist: use of various
types of radio frequencies can alter singlet-to-triplet interconversion of radical-pairs and
disrupt orientation in birds and turtles [67,98,101,107]. To my knowledge, radio-frequency
field experiments have yet to be completed when testing fish magnetoreception. If
application of radio frequency fields disrupted bimodal orientation or a trained magnetic
response, it would provide substantial evidence that zebrafish use radical-pair
65
magnetoreception. These experiments, combined with UV cone ablation, would be a
powerful way to test if cry4 expressing UV cones mediated LDRPM. However, ablation of a
whole photoreceptor subtype is bound to have other effects on visual processing and could
potentially make it difficult to define changes in behavior as a specific loss of
magnetoreception. Targeted mutagenesis is another powerful alternative that can be used
to directly test the requirement of cry in zebrafish magnetoreception.
3.3.2 Genome editing of cry in zebrafish
Zebrafish are amendable to next generation genome editing techniques such as
targeted mutagenesis via the Clustered Regularly Interspaced Short Palindromic Repeat
(CRISPR) and CRISPR associated protein (Cas) system. Cas is a nuclease that digests DNA and
is used in the prokaryotic immune system to protect against foreign genetic material [252]
This function has been exploited to edit genomes of model organisms [253] and is done by
generating a synthetic guide RNA (sgRNA) specific to the gene of interest. Co-injecting the
sgRNA with Cas protein (such as Cas9) into a single-cell staged zebrafish will cause Cas and
the sgRNA to make a complex and bind the target sequence [209]. Once it binds to the specific
area it is designed to, Cas will cut the DNA. Normally the DNA will attempt to repair itself via
non-homologous end joining, which ultimately introduces deletions or insertions into the
target sequence.
Disrupting cry4 and showing a concerted decline in magnetoreceptive abilities would
be a powerful way to show cry4 is necessary for magnetoreception. A caveat with whole
66
organism gene silencing is disrupting the genes endogenous function can have unattended
effects in other tissues it is expressed in. Additionally, although knockout of cry would create
evidence that it is required for magnetoreception, it does not directly point to what sensory
system is being exploited for this behavior. For example, besides retinal expression and
specific localization to UV cones, cry4 is expressed largely in the developing zebrafish brain
and intestine [118,119]. Knockout of cry4 in these tissues may alter normal zebrafish
development, and cause disruption in other cellular processes that may result in a general
behavioral decline, that could be misinterpreted as a loss of magnetoreception. Ideally, cry4
would be manipulated specifically in the cells thought necessary for magnetoreception (such
as UV cones). Tissue specific gene knockout using CRISPR has been developed in zebrafish
[253,254] and will be extremely important when testing the potential role of cry4 and cones
in magnetoreception. To utilize this, one creates a sgRNA targeted to bind somewhere in the
cry4 gene. Slightly upstream of the FAD-binding domain would be ideal, to increase the
chance that the introduced mutation would inhibit the translation of a functional protein.
Then, using a cone specific promoter such as crx (for early development), gnat2 (for post-
mitotic cones) [255] or sws1 (for UV cones) and Tol2 transposase technology (where
transgenes can be inserted into the genome of an animal; [256]), a sgRNA seeding clone can
be made that will drive expression of the sgRNA in the tissue specified by the promoter of
interest (cones). After injection of the sgRNA construct, Tol2 mRNA, and Cas, one can confirm
integration into the genome, and cutting with tissue specific sequencing/ genotyping assays
[209].
cry zebrafish mutants will be important to test if behavioral outputs are changed but
will be particularly useful to ask if cry is required for neuronal processing of magnetic
67
information. Measuring neural activity in response to magnetic stimuli is especially
important in systems that may have multiple modes of magnetoreception. For example, if cry
knockout doesn’t change behavioral outputs, another light-independent mechanism may be
compensating. Neuronal recordings could potentially detect minute changes in the way
magnetic fields are being processed in the presence or absence of cry, and ultimately provide
clues to the neuronal area required for fish magnetoreception.
3.3.3 Imaging magnetically dependent neuronal activity via Ca2+
Standard recording techniques such as electrophysiology are not applicable for
magnetoreception experiments since altering static magnetic fields usually requires running
current through copper wires in a coil system [257]. Due to this, there is potential for
electrical noise to confound recording outputs [175]. New, non-invasive live-imaging
techniques appear to be more suited to test magnetoreception in fish. Genetically encoded
calcium indicators (GECIs) consist of fluorescent molecules fused to proteins that bind Ca2+
such as calmodulin [258]. Activation of neurons normally cause cellular changes of Ca2+
which can be visualized by changes in fluorescence intensity of GECIs. Using this, one could
manipulate experimental magnetic fields and observe neuronal changes in free swimming
zebrafish. This technique could be combined with either: a) targeted cone ablation to test
the role of UV cones in magnetic processing and/or b) UV cone specific knockout of cry4 to
test its role in magnetic detection. Here, it would be predicted that under natural conditions
(unaltered retina), retinal recipients involved in visual processing such as the optic tectum
would show increased activity when magnetic fields are manipulated. Other, non-visual
68
areas that receive input from the retina such as the anterior thalamus [259], may also show
increased activation during magnetic experiments. Retinal-thalamic pathways in zebrafish
may be particularly interesting because the thalamofugal pathway in birds is thought to
mediate magnetoreception ([84,260]. Alteration of magnetic fields after UV cone ablation
may show specific neuronal areas dedicated for processing LDRPM mediated information.
For now, neuronal areas dedicated for magnetoreception remain unknown in zebrafish, and
could potentially be part of a yet to be described circuit.
Our lab has already begun to engineer a GECI in zebrafish by using Calcium-
Modulated Photoactivatable Ratiometric Integrators (CaMPARI) [212]. When CaMPARI is
expressed in all neurons (via the elavl3 promoter), the presence of high intercellular Ca2+
and a photoconversion light causes an irreversible change in the emitted wavelength of a
fluorescent protein from green to red [212]. A caveat here is the use of the photoconversion
laser; its wavelength (405 nm) theoretically could activate UV cones, blue cones and various
intermediates of Cry. By turning the laser on to photoconvert, it is likely that neuronal areas
that receive information from short-wavelength cones and/or Cry will show Ca2+ changes,
regardless of magnetic information. It would be interesting to see if the presence of magnetic
fields showed more activation, or slightly altered activation patterns across the zebrafish
retina and brain after manipulation of the magnetic field. It is likely that if magnetic
information is processed in the same area as visual information is integrated, such as the
optic tectum, CaMPARI will be unable to separate these two signals. For this reason,
traditional GECIs may be more useful for magnetoreception experiments.
69
Another potential issue with using this imaging method and other GECIs in the retina
is the presence of the retinal pigmented epithelium (RPE). Under natural conditions, melanin
deposits in the RPE block visualization of fluorescent molecules in photoreceptors. Using the
CRISPR-Cas9 system, I generated a zebrafish mutant that has a non-pigmented RPE by
knocking out a melanin synthesizing gene slc45a2 (Appendix A; [261]). I crossed slc45a2
mutants to fish with no melanophores in the body (nacre) [262] and no iridophores (roy)
[263] to create a pigmentless zebrafish line (crystal) to improve fluorescent imaging
potential [261]. Another benefit of using crystal is that they do not require treatment with a
common melanin synthesis inhibitor, PTU, which is used to prevent pigment formation. This
drug has been shown to alter various aspects of development including eye formation,
making it non-ideal for experiments testing visually mediated behaviors [264,265].
Furthermore, Ca2+ imaging in the retina of crystal fish has been demonstrated during
exposure to visual stimuli [261], but recordings after magnetic changes have yet to be
described. Currently, CaMPARI in crystal mutants is being established in our lab. GECIs
provide a promising way to visualize magnetically dependent retinal activity in zebrafish.
3.4 Final Conclusions
Magnetoreception has been demonstrated in an overwhelming number of organisms across
a variety of taxa but continues to be one of the remaining senses without an identified
receptor. LDRPM via Cry is the leading hypothesis for magnetoreception in migratory birds
but cannot easily be tested at the molecular level due to the genetic inaccessibility of the
70
avian model. The retina, specifically cone photoreceptors, harbors magnetoreceptive Cry
and may allow the alteration of visual perception by magnetic fields. This thesis work
provides the first evidence that zebrafish have cry in retinal cone photoreceptors.
Specifically, cry4 mRNA is localized in UV cones in both larval and adult zebrafish. Using
targeted cone ablation, I thoroughly validated this association: ablation of UV cones but not
neighboring blue cones, decreased cry4 in zebrafish retina. Paralogous cry2 was undisrupted
after UV and blue cone ablation, supporting that cry4 is specifically associated with UV cones.
Moving forward, a multi-faceted approach will be key in elucidating the mysteries of
magnetoreception. Optically transparent imaging with retinal GECIs, molecular
manipulations via cone ablation or cry knockout and environmental control through
magnetic and radio frequency fields can all be achieved with zebrafish. These highly versatile
vertebrates will be crucial in advancing our understanding of this complex phenomenon.
71
Figure 12. Molar absorption spectra of FAD in different Cry redox states. Spectra
reused with permission from Bazalova et al., [14]. FADox originally from Islam et al., [223];
FAD•− and FADH• from Palfey and Massey [222]; FADH− from Muller [225].
72
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APPENDIX A
A.1 Generation of slc45a2 mutant zebrafish
I aimed to create a zebrafish mutant that lacked melanin in the RPE to allow for better
visualization of fluorescent reporters in zebrafish photoreceptors. To do this, I utilized
CRISPR-Cas9 genome editing based on [209].
A.1.1 sgRNA creation
sgRNA synthesis was completed as previously described [209]. Briefly, gene-specific
oligonucleotides designed after [266] containing the SP6 promoter sequence, the 20-base
pair target site without the Protospacer adjacent motif (PAM) sequence, and a
complementary region were annealed to a constant oligo (Table A1). ssDNA was filled with
T4 DNA polymerase (NEB, M0203S). sgRNA template was purified using QIAquick PCR
Purification Kit (Qiagen, 28104) and transcribed using mMESSAGE mMACHINE™ SP6
Transcription Kit (Invitrogen, AM1340). sgRNA was treated with DNAse I (Qiagen, 79254)
and quantified using Nanodrop spectrophotometer (GE Healthcare 28 9244-02). sgRNA was
also run out on a 3% agarose gel to confirm transcription.
A.1.2 Injection process
Zebrafish nacre strain (mitfa-/-) were injected with 2-3 nl of injection solution (5 µl: 2 µl ~200
ng/µl sgRNA, 2 µl of 20 µM Cas9, 0.5 µl 1x Cas9 Nuclease Buffer (NEB, M0646M), 0.5 µl phenol
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red into the cell of one-cell staged embryos. Embryos were grown and screened for pigment
defects in the RPE at 3-4 days post fertilization (dpf) (Figure A1). Fish with pigment
alterations were grown to adulthood.
A.1.3 Genotyping via sequencing and RFLP
Adult zebrafish pigment mutants were fin clipped and genomic DNA (gDNA) was extracted
as previously described [267]. PCR primers (Table A2) were used to amplify the CRISPR site
target region. Template was cloned into pCR 2.1 plasmids via TA TOPO cloning (Invitrogen,
450641) and transformed. Colonies were picked and cultured in LB media, and plasmid DNA
was isolated via QIAprep Spin Miniprep Kit (Qiagen, 27104). Plasmids were sequenced at
the Molecular Biology Services Unit at the University of Alberta with amplicon specific
primers (Table A2) to identify mutations.
A 22 base-pair (bp) deletion mutation was identified in fish with pigment defects, which
causes a frameshift and early STOP codon at amino acid position 447 in SLC45A2 (Figure
A1E). These mutants (ua5015; slc45a2-/-; mitfa-/-) were subsequently outcrossed to nacre
background (slc45a2+/+; mitfa-/-) to determine germline transmission. Heterozygotes for
ua5015 were identified by restriction fragment length polymorphism analysis (RFLP).
Primers were designed to amplify a 235 bp region including the ua5015 scl45a2 locus (Table
A2). PCR product was digested with MspI restriction enzyme (NEB, R0106S) and run on a
gel to identify wild-type, heterozygous or homozygous ua5015 alleles.
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A.1.4 Generation of crystal and crystal-based transgenics
To generate crystal mutants [261], ua5015 were crossed to casper mutants (slc45a2+/+;
nacre-/-; roy-/-) [263], genotyped as described above and in-crossed to create our crystal line
(ua5020; slc45a2-/-; nacre-/-; roy-/- ) (Figure A4).
u5020 was outcrossed to Tg(sws1:nfsb-mCherry; sws2:GFP) in a roy-/- background [174,179],
genotyped and in-crossed as described to create Tgua5020(sws1:nfsb-mCherry; sws2:GFP)
(Figure A5) which allow fluorescent visualization of UV and blue cones. Additionally, UV
cones can be ablated in this line. Overall, Tgua5020(sws1:nfsb-mCherry; sws2:GFP) combined
with GECI technology can be used to determine the role of UV cones in neuronal processing
of magnetic information.
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Table A1. Oligonucleotides used for sgRNA synthesis.
Table A2. Primers used for ua5015 and ua5020 genotyping.
Oligo Oligo sequences (SP6 promoter, slc45a2 CRISPR site, overlap region for annealing)
slc45a2 sgRNA
ATTTAGGTGACACTATAGGTTTGGGAACCGGTCTGATGTTTTAGAGCTAGAAATA
GCAAG
Constant oligo
AAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTAT
TTTAACTTGCTATTTCTAGCTCTAAAAC
Gene and Primer Use Amplicon Size (bp)
Primer sequences (forward, reverse)
slc45a2; ua5015 TOPO cloning and sequencing
733 F: 5’ ATTGTATATAAGGGGAATCCGTATGCT ‘3
R: 5’ CTCTTCTGCCTTGTGGTACTC ‘3
slc45a2; ua5015 RFLP
235 F: 5’ TTCATCCATTTGTTCTGCATTAAAGGC ‘3
R: 5’ GAGTCATGTCCAGCACTCTCTACAC ‘3
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Figure A1. Larval nacre zebrafish injected with slc45a2 CRISPR mix show mosaic
pigment defects. A) Uninjected sibling with large deposits of melanin in the RPE B-B’)
Various mosaic pigment phenotypes are observed when nacre zebrafish are injected with
slc45a2 CRISPR mix. Larvae observed at 3 days post fertilization (dpf). RPE= retinal
pigmented epithelium.
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Figure A2. ua5015 (slc45a2-/-; mitfa-/-) have no pigment in the RPE and can be observed
through development. nacre fish have a pigmented RPE at A-C) 3dpf and D) 4 months while
ua5015 fish have transparent RPE’s at A’-C’) 3dpf and D’) 4 months. E) ua5015 is defined by
a 22 base pair deletion in slc45a2. ZFIN CRISPR1 from [266].
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98
Figure A3. Pigmentless ua5020 mutants are generated from crossing ua5015 to
casper. A) Close up of nacre eye, showing iridophores around the eye and melanin in the
RPE. A’) Iridophores in body of nacre can be seen. B) Close up of ua5015 eye, showing
iridophores but no melanin in the RPE due to mutation in slc45a2. B’) Iridophores in body
of ua5015 can still be observed. C) Close up of casper eye, showing no iridophores due to a
mutation in roy. Melanin can still be seen in the RPE. C’) Iridophores and melanophores are
absent in body of casper. D) Close up of ua5020 eye, showing no iridophores and no
melanin in RPE. D’) Iridophores and melanophores are absent in body of ua5020.
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Figure A4. Tgua5020(sws1:nfsb-mCherry; sws2:GFP) transgenics allow easy visualization
of fluorescent reporters in zebrafish retina. A) Dorsal view of adult (4 months old)
ua5020 head in brightfield. Transparent eyes can be seen on either side. B) mCherry marks
nitroreductase in UV cones and C) GFP marks blue cones. D) Overlay of both channels, E) the
mCherry channel and F) the GFP channel on a dorsal brightfield image.